Therapeutic pharmaceutical compositions

ABSTRACT

Provided herein are pharmaceutical compositions comprising a bacterial consortium and one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can be orally administered to a subject for prevention and/or treatment of dysbiosis, dysbiosis associated conditions, inflammation, and autoimmune diseases.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.17/475,228, filed Sep. 14, 2021, which is a continuation ofInternational Patent Application No. PCT/US2020/054444, filed Oct. 6,2020, which claims priority to and the benefit of U.S. ProvisionalApplication No. 62/911,873, filed Oct. 7, 2019, the entire contents ofeach of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 2, 2020, isnamed 53206-707_601_SL.txt and is 3,713 bytes in size.

BACKGROUND

Recent developments in the areas of microbiome and genome researchprovide evidence that the bacterial composition of the human gutfundamentally influences human health, disease onset and progression.However, much remains unknown with regards to the microbiome-hostrelationships, functional and metabolic changes in host due to themicrobiome composition, as well as the potential development ofbacterial compositions for therapeutic applications. For example, andalthough there is an established genetic component contributing to riskfor inflammatory diseases such as allergy and asthma, environmentalfactors including microbial exposure and microbiome composition play keyroles in the etiology of these disease. Data from clinical trials showthat the gut microbiomes of infants who eventually develop allergy andasthma are distinct in bacterial species composition andimmunomodulatory activity. The rising incidence of allergic diseases ingeneral is concerning, and asthma is worrisome because it represents arapidly growing global concern. As there is no cure for either of theseallergic diseases, they represent a major public health challenge withbillions of dollars spent managing their symptoms. Asthma, for example,carries a steep cost burden of over 56 billion dollars per year in theUS alone, of which nearly 20 billion dollars are spent on standard ofcare treatments that have variable efficacy and potentially serious sideeffects. As a result, expansion of treatment options, such asimmunomodulatory microbial compositions targeting allergic diseaseprevention, would address a serious unmet need, especially indisproportionately affected pediatric populations.

BRIEF SUMMARY

Provided herein are pharmaceutical compositions in solid dosage formsand comprises: a purified bacterial population comprising at least onestrain of Akkermansia sp., Faecalibacterium sp., and Lactobacillus sp.;and a cryoprotectant. Further provided herein is a pharmaceuticalcomposition, wherein the cryoprotectant comprises a carbohydrate and anantioxidant. Further provided herein is a pharmaceutical composition,wherein the carbohydrate comprises a saccharose, a trehalose, or acombination thereof. Further provided herein is a pharmaceuticalcomposition, wherein the antioxidant comprises an amino acid. Furtherprovided herein is a pharmaceutical composition, wherein the amino acidcomprises a L-glutamate, a L-cysteine, or a combination thereof. Furtherprovided herein is a pharmaceutical composition, wherein the L-cysteineis presented, by weight, in an amount from about 0.05% to about 1%.Further provided herein is a pharmaceutical composition, wherein thecryoprotectant comprises, by weight, about 60% saccharose, about 10%trehalose, about 1% L-cysteine, and about 4% L-glutamate. Furtherprovided herein is a pharmaceutical composition, wherein thepharmaceutical composition is formulated into a suspension. Furtherprovided herein is a pharmaceutical composition, wherein thepharmaceutical composition is formulated as an oral dosage form. Furtherprovided herein is a pharmaceutical composition, wherein the oral dosageform is a capsule, tablet, emulsion, suspension, syrup, gel, gum, paste,herbal tea, drops, dissolving granules, powders, tablets, lyophilizate,a popsicle, or ice cream. Further provided herein is a pharmaceuticalcomposition, wherein the bacterial population is lyophilized.

Provided herein are pharmaceutical compositions in liquid dosage formsand comprises: a purified bacterial population comprising at least onestrain of Akkermansia sp., Faecalibacterium sp., and Lactobacillus sp.;and an antioxidant. Further provided herein is a pharmaceuticalcomposition, further comprises a cryoprotectant. Further provided hereinis a pharmaceutical composition, wherein the cryoprotectant comprisesglycerol. Further provided herein is a pharmaceutical composition,wherein the glycerol is present, by volume, in an amount from about 10%to about 30%. Further provided herein is a pharmaceutical composition,wherein the antioxidant comprises an amino acid. Further provided hereinis a pharmaceutical composition, wherein the amino acid comprises anL-cysteine. Further provided herein is a pharmaceutical composition,wherein the L-cysteine is present, by weight, in an amount of about0.1%. Further provided herein is a pharmaceutical composition, furthercomprising a buffer. Further provided herein is a pharmaceuticalcomposition, wherein the buffer is phosphate buffered saline (PBS) andhas a pH of about 7.4. Further provided herein is a pharmaceuticalcomposition, wherein the pharmaceutical composition has a total volumeof about 1 mL. Further provided herein is a pharmaceutical composition,further comprises a container. Further provided herein is apharmaceutical composition, wherein the container is a 2 mLpolypropylene screw cap vial.

Provided herein are pharmaceutical compositions, comprising: a purifiedbacterial population comprising at least one strain of Akkermansia sp.,Lactobacillus sp., and Faecalibacterium sp.; and a pharmaceuticallyacceptable excipient, wherein the pharmaceutical composition isencompassed in a capsule, and wherein the capsule comprises aplant-derived material. Further provided herein is a pharmaceuticalcomposition, wherein the plant-derived material comprises acellulose-based polymer. Further provided herein is a pharmaceuticalcomposition, wherein the cellulose-based polymer comprises pullulan.Further provided herein is a pharmaceutical composition, wherein thecapsule has an oxygen permeability less than about 0.5 cm³/m²/day, asmeasured by a gas composition in the capsule. Further provided herein isa pharmaceutical composition, wherein the capsule has a disintegrationendpoint of about 1.6 minutes, as measured at 37° C. with de-ionizedwater. Further provided herein is a pharmaceutical composition, furthercomprises a cryoprotectant. Further provided herein is a pharmaceuticalcomposition, wherein the cryoprotectant comprises a carbohydrate and anantioxidant. Further provided herein is a pharmaceutical composition,wherein the bacteria population is lyophilized. Further provided hereinis a pharmaceutical composition, wherein the at least one strain ofAkkermansia sp., Faecalibacterium sp., and Lactobacillus sp. is selectedfrom the strains listed in TABLE 1. Further provided herein is apharmaceutical composition, wherein the bacterial population comprisesA. muciniphila (DSM 33213), F. prausnitzii (DSM 33185), or L. crispatus(DSM 33187). Further provided herein is a pharmaceutical composition,wherein the bacterial population comprises at least two of the bacterialstrains A. muciniphila (DSM 33213), F. prausnitzii (DSM 33185), and L.crispatus (DSM 33187). Further provided herein is a pharmaceuticalcomposition, wherein the bacterial population comprises the bacterialstrains A. muciniphila (DSM 33213), F. prausnitzii (DSM 33185), and L.crispatus (DSM 33187). Further provided herein is a pharmaceuticalcomposition, wherein each bacterial strain is present in an amount fromabout 10{circumflex over ( )}3 CFU to about 10{circumflex over ( )}12CFU. Further provided herein is a pharmaceutical composition, whereineach bacterial strain is present in an amount from about 10{circumflexover ( )}7 CFU to about 10{circumflex over ( )}10 CFU. Further providedherein is a pharmaceutical composition, wherein each bacterial strain ispresent in an amount of about 5×10{circumflex over ( )}8 CFU. Furtherprovided herein is a pharmaceutical composition, wherein the bacterialpopulation is present in a total amount of about 10{circumflex over( )}3 CFU to about 10{circumflex over ( )}12 CFU. Further providedherein is a pharmaceutical composition, wherein the bacterial populationis present in a total amount of about 10{circumflex over ( )}7 CFU toabout 10{circumflex over ( )}10 CFU. Further provided herein is apharmaceutical composition, wherein the bacterial population is presentin a total amount of about 1.5×10{circumflex over ( )}9 CFU.

Provided herein are method of treating a disease in a subject, themethod comprising administering to the subject any pharmaceuticalcomposition previously described. Further provided herein is a methodfor treating a disease in a subject, wherein the disease is aninflammatory disease. Further provided herein is a method for treating adisease in a subject, wherein the inflammatory disease is an allergy ordermatitis. Further provided herein is a method for treating a diseasein a subject, wherein the allergy is allergic asthma, allergic pediatricasthma or food allergy. Further provided herein is a method for treatinga disease in a subject, wherein the disease is a metabolic disease.Further provided herein is a method for treating a disease in a subject,wherein the metabolic disease is obesity, diabetes, or a metabolicsyndrome.

Provided herein is methods of reducing the incidence of an allergiccondition in a subject, the method comprising orally administering tothe subject a pharmaceutical composition comprising a purified bacterialpopulation comprising a strain of Akkermansia sp., Faecalibacterium sp.,and Lactobacillus sp., wherein the pharmaceutical composition isadministered to the subject at least once daily for at least 7 days.Further provided herein is a method of reducing the incidence of anallergic condition in a subject, wherein the subject is a neonate ofequal to or less than about 7 days of age. Further provided herein is amethod of reducing the incidence of an allergic condition in a subject,wherein the subject is an infant of from about 28 days to about 12months of age. Further provided herein is a method of reducing theincidence of an allergic condition in a subject, wherein thepharmaceutical composition is administered to the subject for at least28 days. Further provided herein is a method of reducing the incidenceof an allergic condition in a subject, wherein the pharmaceuticalcomposition is administered to the subject for at least 336 days.Further provided herein is a method of reducing the incidence of anallergic condition in a subject, wherein the pharmaceutical compositionis administered to the subject once, two, three, four, five, or sixtimes daily. Further provided herein is a method of reducing theincidence of an allergic condition in a subject, wherein thepharmaceutical administered to the subject once, two, three, four, five,six, seven, eight, nine, ten, eleven, or twelve times daily. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the allergic condition is atopicdermatitis, food allergy, allergic rhinitis, or allergic asthma. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the subject has a biological mother,father, or sibling with a history of an allergic condition is atopicdermatitis, food allergy, allergic rhinitis, or allergic asthma. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the subject has a birthweight from about2.5 kg to 4.5 kg. Further provided herein is a method of reducing theincidence of an allergic condition in a subject, wherein each bacterialstrain is present in an amount from about 10{circumflex over ( )}3 CFUto about 10{circumflex over ( )}12 CFU. Further provided herein is amethod of reducing the incidence of an allergic condition in a subject,wherein each bacterial strain is present in an amount from about10{circumflex over ( )}7 CFU to about 10{circumflex over ( )}10 CFU.Further provided herein is a method of reducing the incidence of anallergic condition in a subject, wherein each bacterial strain ispresent in an amount of about 5×10{circumflex over ( )}8 CFU. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the bacterial population is present in atotal amount of about 10{circumflex over ( )}3 CFU to about10{circumflex over ( )}12 CFU. Further provided herein is a method ofreducing the incidence of an allergic condition in a subject, whereinthe bacterial population is present in a total amount of about10{circumflex over ( )}7 CFU to about 10{circumflex over ( )}10 CFU.Further provided herein is a method of reducing the incidence of anallergic condition in a subject, wherein the bacterial population ispresent in a total amount of about 1.5×10{circumflex over ( )}9 CFU.Further provided herein is a method of reducing the incidence of anallergic condition in a subject, wherein the Akkermansia sp.,Faecalibacterium sp., and Lactobacillus sp. are administered at an equalamount. Further provided herein is a method of reducing the incidence ofan allergic condition in a subject, wherein the Akkermansia sp.,Faecalibacterium sp., and Lactobacillus sp. are administered at5×10{circumflex over ( )}8 CFU each. Further provided herein is a methodof reducing the incidence of an allergic condition in a subject, furthercomprising a carbohydrate-based excipient. Further provided herein is amethod of reducing the incidence of an allergic condition in a subject,wherein the pharmaceutical composition is mixed into breast milk,formula, or food. Further provided herein is a method of reducing theincidence of an allergic condition in a subject, wherein the suspensioncomprises a cryoprotectant. Further provided herein is a method ofreducing the incidence of an allergic condition in a subject, whereinthe cryoprotectant comprises glycerol. Further provided herein is amethod of reducing the incidence of an allergic condition in a subject,wherein the glycerol is present, by volume, in an amount from about 10%to about 30%. Further provided herein is a method of reducing theincidence of an allergic condition in a subject, further comprises anantioxidant. Further provided herein is a method of reducing theincidence of an allergic condition in a subject, wherein the antioxidantcomprises an amino acid. Further provided herein is a method of reducingthe incidence of an allergic condition in a subject, wherein the aminoacid comprises an L-cysteine. Further provided herein is a method ofreducing the incidence of an allergic condition in a subject, whereinthe L-cysteine is present, by weight, in an amount of about 0.1%.Further provided herein is a method of reducing the incidence of anallergic condition in a subject, further comprising a buffer. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the buffer is phosphate buffered saline(PBS) and has a pH of about 7.4. Further provided herein is a method ofreducing the incidence of an allergic condition in a subject, whereinthe pharmaceutical composition has a total volume of about 1 mL. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the composition is encompassed in acapsule. Further provided herein is a method of reducing the incidenceof an allergic condition in a subject, wherein the capsule comprisesplant-based material. Further provided herein is a method of reducingthe incidence of an allergic condition in a subject, wherein the capsulecomprises cryoprotectant. Further provided herein is a method ofreducing the incidence of an allergic condition in a subject, whereinthe cryoprotectant comprises a carbohydrate and an antioxidant. Furtherprovided herein is a method of reducing the incidence of an allergiccondition in a subject, wherein the carbohydrate comprises a saccharose,a trehalose, or a combination thereof. Further provided herein is amethod of reducing the incidence of an allergic condition in a subject,wherein the antioxidant comprises an amino acid. Further provided hereinis a method of reducing the incidence of an allergic condition in asubject, wherein the amino acid comprises a L-glutamate, a L-cysteine,or a combination thereof. Further provided herein is a method ofreducing the incidence of an allergic condition in a subject, whereinthe L-cysteine is presented, by weight, in an amount from about 0.05% toabout 1%. Further provided herein is a method of reducing the incidenceof an allergic condition in a subject, wherein the cryoprotectantcomprises, by weight, about 60% saccharose, about 10% trehalose, about1% L-cysteine, and about 4% L-glutamate. Further provided herein is amethod of reducing the incidence of an allergic condition in a subject,wherein the bacterial population is lyophilized.

Provided herein are methods for a large-scale growth of Akkermansia sp.comprising performing a plurality of inoculation rounds with anincreasing amount of growth media, wherein each inoculation roundcomprises at least about 5% of a total batch material of a precedinginoculation round. Further provided herein is a method for a large-scalegrowth of Akkermansia sp., wherein the Akkermansia sp. comprisesAkkermansia muciniphila or Akkermansia glycaniphila. Further providedherein is a method for a large-scale growth of Akkermansia sp., whereinthe Akkermansia muciniphila comprises Akkermansia muciniphila (DSM33213). Further provided herein is a method for a large-scale growth ofAkkermansia sp., wherein the growth media is from about 1 L to about4,000 L. Further provided herein is a method for a large-scale growth ofAkkermansia sp., further comprising an initial inoculation round ofabout 1 L growth media. Further provided herein is a method for alarge-scale growth of Akkermansia sp., wherein at least one of theinoculation rounds is in a volume of at least about 3000 L growth media.Further provided herein is a method for a large-scale growth ofAkkermansia sp., wherein the initial inoculation round comprises afrozen stock of Akkermansia muciniphila of about 2% of an initialinoculation round growth media. Further provided herein is a method fora large-scale growth of Akkermansia sp., wherein the initial inoculationround comprises growing Akkermansia muciniphila in anaerobic condition.Further provided herein is a method for a large-scale growth ofAkkermansia sp., further comprising a final inoculation round, whereinthe final inoculation round comprises Akkermansia sp. present in anamount of OD600 of at least 2.5. Further provided herein is a method fora large-scale growth of Akkermansia sp., further comprising a finalinoculation round of, by volume, about 10% of the total batch materialof the preceding inoculation round. Further provided herein is a methodfor a large-scale growth of Akkermansia sp., further comprisingperforming a plurality of sterilization rounds for the growth media,wherein each sterilization round comprises degassing the growth mediawith N₂H₂CO₂ (90:5:5). Further provided herein is a method for alarge-scale growth of Akkermansia sp., further comprising lyophilizingthe batch. Further provided herein is a method for a large-scale growthof Akkermansia sp., further comprising centrifuging the batch before thelyophilizing. Further provided herein is a method for a large-scalegrowth of Akkermansia sp., further comprising grinding the batch afterthe lyophilizing. Further provided herein is a method for a large-scalegrowth of Akkermansia sp., wherein during the period of growth of eachinoculation round, the growth media has a pH value of less than about 7.Further provided herein is a method for a large-scale growth ofAkkermansia sp., wherein during the period of growth of each inoculationround, the growth media has a pH value of less than about 6.5.

Provided herein are methods for a large-scale growth of Faecalibacteriumsp. comprising performing a plurality of inoculation rounds with anincreasing amount of growth media, wherein during the period of growthof each inoculation round, the growth media has a pH value of less thanabout 6.5. Further provided herein is a method for a large-scale growthof Faecalibacterium sp., wherein the Faecalibacterium sp. comprisesFaecalibacterium prausnitzii. Further provided herein is a method for alarge-scale growth of Faecalibacterium sp., wherein the Faecalibacteriumsp. comprises Faecalibacterium prausnitzii. Further provided herein is amethod for a large-scale growth of Faecalibacterium sp., whereinFaecalibacterium prausnitzii comprises Faecalibacterium prausnitzii (DSM33185). Further provided herein is a method for a large-scale growth ofFaecalibacterium sp., wherein the growth media is from about 1 L toabout 4,000 L. Further provided herein is a method for a large-scalegrowth of Faecalibacterium sp., further comprising an initialinoculation round of about 1 L growth media. Further provided herein isa method for a large-scale growth of Faecalibacterium sp., wherein atleast one of the inoculation rounds is at least about 3000 L growthmedia. Further provided herein is a method for a large-scale growth ofFaecalibacterium sp., wherein the initial inoculation round comprises afrozen stock of Faecalibacterium prausnitzii of about 0.4% of an initialinoculation round growth media. Further provided herein is a method fora large-scale growth of Faecalibacterium sp., wherein the initialinoculation round comprises growing Faecalibacterium prausnitzii inanaerobic condition. Further provided herein is a method for alarge-scale growth of Faecalibacterium sp., further comprising a finalinoculation round, wherein the final inoculation round comprisesFaecalibacterium sp present in an amount of OD₆₀₀ of at least 5. Furtherprovided herein is a method for a large-scale growth of Faecalibacteriumsp., further comprises performing a plurality of sterilization anddegassing rounds for the growth media, wherein each sterilization roundcomprises autoclaving the growth media at 121° C. for 20 minutes, andwherein each degassing round comprises degassing the growth media withN₂H₂CO₂ (90:5:5). Further provided herein is a method for a large-scalegrowth of Faecalibacterium sp., further comprising lyophilizing thebatch. Further provided herein is a method for a large-scale growth ofFaecalibacterium sp., further comprising centrifuging the batch beforethe lyophilizing. Further provided herein is a method for a large-scalegrowth of Faecalibacterium sp., further comprising grinding the batchafter the lyophilizing. Further provided herein is a method for alarge-scale growth of Faecalibacterium sp., wherein during the period ofgrowth of each inoculation round, the growth media has a pH value ofless than about 6. Further provided herein is a method for a large-scalegrowth of Faecalibacterium sp., wherein during the period of growth ofeach inoculation round, the growth media has a pH value of less thanabout 5.5. Further provided herein is a method for a large-scale growthof Faecalibacterium sp., wherein during the period of growth of eachinoculation round, the growth media has a pH value of less than about 5.Further provided herein is a method for a large-scale growth ofFaecalibacterium sp., wherein each inoculation round comprises at leastabout 1% of a total batch material of a preceding inoculation round.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (“FIGURE.” or “FIGURES.” herein), of which:

FIG. 1 shows a schematic flow chart summarizing the manufacturing stepsof compositions as described herein.

FIG. 2 shows a chart of light absorption at 600 nanometer (also referredto herein as “OD₆₀₀”) wavelength versus time for measurement of growthover time for A. muciniphila in modified NAGT media. The data is fromvariation in media that includes: unmodified NAGT media, modified NAGTmedia, modified NAGT media with no glucose, modified NAGT media with nocalcium and modified NAGT media with no magnesium.

FIG. 3 shows a chart of light absorption at 600 nanometer wavelengthversus time for measurement of growth over time for A. muciniphila indifferent NAGT media. The data is from variation in media that includes:unmodified NAGT media, NAGT with no NAG, and NAGT with no soytone.

FIG. 4 shows a chart of light absorption at 600 nanometer wavelengthversus time for measurement of growth over time for F. prausnitzii indifferent growth media. The data is from variation in media thatincludes: complete media, media with no sodium acetate, media with nosoytone, media with no yeast extract, and media with no cysteine.

FIG. 5 shows a chart of light absorption at 600 nanometer wavelengthversus time for the growth of F. prausnitzii (DSM 33185) in YFAP mediawith different supplements. F. prausnitzii was sub-cultured 3 times inYFAP without vitamin before growing in YFAP media with differentsupplements. The data is from variation in media that includes YFAPmedia with complete vitamin mix (Vitamin Mix Solution) (YFAP+vitamin),YFAP media without biotin (YFAP No biotin), YFAP media without cobalamin(YFAP No cobalamin), YFAP media without PABA (YFAP No PABA), YFAP mediawithout folic acid (YFAP No folic acid), YFAP media without pyridoxamine(YFAP No pyridoxamine), YFAP media without thiamine (YFAP No thiamine),YFAP media without riboflavin (YFAP No riboflavin), and YFAP mediawithout complete vitamin mix (YFAP No vitamin). Addition of vitaminincreased the growth of F. prausnitzii by about 10%.

FIG. 6 shows a chart of light absorption at 600 nanometer wavelengthversus time for the growth of F. prausnitzii (DSM 33185) in YFAP mediawith different supplements. F. prausnitzii was sub-cultured 1 timewithout vitamin. The data is from variation in media that includes: YFAPmedia with complete vitamin mix (Vitamin Mix Solution) (YFAP+vitamin),YFAP media without biotin (YFAP No biotin), YFAP media without cobalamin(YFAP No cobalamin), YFAP media without PABA (YFAP No PABA), YFAP mediawithout folic acid (YFAP No folic acid), YFAP media without pyridoxamine(YFAP No pyridoxamine), YFAP media without thiamine (YFAP No thiamine),YFAP media without riboflavin (YFAP No riboflavin), and YFAP mediawithout complete vitamin mix (YFAP No vitamin). The lack of eithercomplete vitamin mix or cobalamin decreased the growth of F. prausnitziiby about 30%.

FIG. 7 shows a chart of light absorption at 600 nanometer wavelengthversus time for the growth of F. prausnitzii (DSM 33185) in YFAP mediawithout pH control. The culture was inoculated at 24^(th) hour, stirredat 100 rpm, and incubated at 37° C. When pH was not fixed, it dropped toabout 5.5. The redox maintained at −400 mV, and the bacterial growthreached OD₆₀₀=1.1. Any reading before 24^(th) hour represents thebaseline value for each measurement.

FIG. 8 shows a chart of light absorption at 600 nanometer wavelengthversus time for the growth of F. prausnitzii (DSM 33185) in YFAP mediawith pH control. The culture was inoculated at 24^(th) hour, stirred at100 rpm, and incubated at 37° C. When pH was fixed by the addition ofammonium hydroxide (NH₄OH) at 6.75, the redox dropped to −460 mV whilethe bacterial growth plateaued at around OD₆₀₀=0.5. Any reading before24^(th) hour represents the baseline value for each measurement.

FIG. 9 shows a chart of light absorption at 600 nanometer wavelengthversus time of the growth of L. crispatus over time in different growthmedia. The chart compares the growth of L. crispatus in Boullion vMRSbroth and HiMedia vMRS broth.

FIG. 10 shows a chart of light absorption at 585 nanometer wavelength(also referred to herein as “OD₅₈₅”) (Y-axis on the left) and glucoseconcentration (g/L) (Y-axis on the right) versus time for the growth ofA. muciniphila (DSM 33213) 20 liter culture in NAGT media. The chartshows the relationship between the growth of A. muciniphila and glucoselevel over time. The addition (*) of glucose (4.52 g/L) andN-acetylglucosamine (5.54 g/L) at 27^(th) and 48^(th) hour allowed theA. muciniphila culture to maintain in the exponential phase between25^(th) to 50^(th) hour and enter the stationary phase after 50^(th)hour

FIG. 11 shows a chart of light absorption at 585 nanometer wavelength(Y-axis on the left) and glucose concentration (g/L) (Y-axis on theright) versus time for the growth of L. crispatus (DSM 33187) 20 literculture in Vegitone MRS media. The chart shows the relationship betweenthe growth of L. crispatus (DSM 33187) and glucose level over time. Thetwo additions (1^(st) feed * and 2^(nd) feed **) of glucose (10 g/L) at10^(th) and 11^(th) hour allowed the L. crispatus culture to maintain inthe exponential phase between 11^(th) to 14^(th) hour and enter thestationary phase after 14^(th) hour.

FIG. 12 shows a chart of light absorption at 585 nanometer wavelength(Y-axis on the left) and glucose concentration (g/L) (Y-axis on theright) versus time for the growth of F. prausnitzii (DSM 33185) 20 literculture in FAP media. The chart shows the relationship between thegrowth of F. prausnitzii (DSM 33185) and glucose level over time. Theaddition (1^(st) feed *) of glucose (10 g/L) at 9^(th) hour allowed theF. prausnitzii culture allow the cell to maintain growth from 9^(th) to14^(th) hour.

FIG. 13 shows a chart of light absorption at 585 nanometer wavelength(Y-axis on the left) and glucose concentration (g/L) (Y-axis on theright) versus time for the growth of A. muciniphila (DSM 33213) 150liter culture in NAGT media. The chart shows the relationship betweenthe growth of A. muciniphila and glucose level over time. The addition(*) of glucose (4.52 g/L) and N-acetylglucosamine (5.54 g/L) at 19^(th)hour allowed the A. muciniphila culture to maintain in the exponentialgrowth after 20^(th) hour.

FIG. 14 shows a chart of light absorption at 585 nanometer wavelength(Y-axis on the left) and glucose concentration (g/L) (Y-axis on theright) versus time for the growth of L. crispatus (DSM 33187) 150 literculture in Vegitone MRS media. The chart shows the relationship betweenthe growth of L. crispatus (DSM 33187) and glucose level over time. Theaddition (*) of glucose (35 g/L) at 10^(th) hour allowed the L.crispatus culture to maintain in the exponential growth from 10^(th) to12^(th) hour and enter stationary growth after 12^(th) hour.

FIG. 15 shows a chart of light absorption at 600 nanometer wavelengthversus time for the measurement of growth over time of the 1 L F.prausnitzii (DSM 33185) culture in YFAP-NU media.

FIG. 16 shows a chart of light absorption at 600 nanometer wavelengthversus time for the measurement of growth over time of the 150 L F.prausnitzii (DSM 33185) culture in YFAP-NU media. The addition (*) ofglucose (10 g/L) at 8^(th) hour allowed the F. prausnitzii culture toenter exponential phase and reach stationary phase.

FIGS. 17A-17C show a flow cytometry gating experiment of heat-killedcontrol A. muciniphila (DSM 33213) cells for quantification ofmetabolically active therapeutic strains in a bacterial cell population,e.g., a cell population that can be administered to a human subject.Used as an example strain, A. muciniphila (DSM 33213) cell stocksolutions were diluted to 10{circumflex over ( )}−4 M in 0.9% NaClbuffer solution and placed in a heating block at 95° C. for 20 minutesto ensure cell death prior to performing the experiment. Cells werestained with 2 μM of propidium iodide and 2 μM of SYTO9. A gate isapplied to all cells (FIG. 17A) counted by Forward Scatter Area (FSC-A)and Side Scatter Area (SSC-A) to select for cell size and granularity,respectively. Those cells were then gated on linearity based on ForwardScatter Height (FSC-H) and Forward Scatter-Area (FSC-A) to identifysingle cells (FIG. 17B). The single cells were then used to set gatesfor dead cells (PIhighSYTO9 low) as well as live cells (PI-SYTO9 high),which gave the percentages of live and dead cells in 50 μl of solution(FIG. 17C).

FIG. 17A shows flow cytometry results obtained when a gate was appliedto all cells counted by Forward Scatter Area (FSC-A) and Side ScatterArea (SSC-A) to select for cell size and granularity, respectively.

FIG. 17B shows flow cytometry results obtained when cells were gated onlinearity based on Forward Scatter Height (FSC-H) and ForwardScatter-Area (FSC-A) to identify single cells.

FIG. 17C shows flow cytometry results obtained when single cells wereused to set gates for dead cells (PIhighSYTO9 low) as well as live cells(PI-SYTO9 high), which gave the percentages of live and dead cells in 50μL of cell suspension.

FIG. 18 shows a graph comparing flow cytometry quantification data oflive cells with the number of total live cells determined using the(standard) agar plating method. The left y-axis shows the number ofTotal Live Cells measured by flow cytometry. The right y-axis shows thecalculated Average CFU/mL values from nutrient agar plating forbiological duplicates. A two tailed Mann Whitney t-test showed nosignificant difference between the mean values of the two quantificationmethods with a p-value of 0.0532.

FIGS. 19A-19C show limit of detection curves (the control thresholdvalue was plotted against the number of bacterial cells) for quantifyingthe bacterial strains Akkermansia muciniphila (DSM 33213) (FIG. 19A),and Faecalibacterium prausnitzii (DSM 33185) (FIG. 19B), andLactobacillus crispatus(DSM 33187) (FIG. 19C), respectively, in a fecalsample. The dotted lines connect the measured data points, the straightlines represent the fitted regression lines, and “R²” is the coefficientof determination. The data shown represent standard curves generatedusing pure bacterial DNA (10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001nanogram (ng) of 50 ng of total DNA (strain DNA+fecal DNA) of the threestrains diluted in fecal DNA background. In instances where humansamples are analyzed to determine the amount of strain (e.g., A.muciniphila (DSM 33213), F. prausnitzii (DSM 33185), and L. crispatus(DSM 33187)) DNA, the control samples can be run and analyzed inparallel to ensure the DNA primers used in the experiments are able toproperly amplify the DNA of the bacterial strains.

FIG. 20 shows a schematic flow chart for an optimized andultra-large-scale growth and manufacturing process for A. muciniphila(DSM 33213) in 3500 L culture volume. 19.2 mL of Working Cell Bank (WCB)A. muciniphila (DSM 33213) was thawed in the anaerobic chamber andinoculated in 1 L of reduced NAGT media (2% v/v inoculation rate) in a 1L bottle 2001 in an anaerobic chamber. The cultured was stopped wheneither OD₅₈₅>1 or the culture grew for 48 hours. The entire culture in2001 was used to inoculate in 16 L media (5% v/v inoculation rate) in a20 L fermenter 2002. The cultured was stopped when either OD₅₈₅>1.5 orthe culture grew for 48 hours. 15 L culture in 2002 was used toinoculate in 300 L media (5% v/v inoculation rate) in a 300 L fermenter2003. The cultured was stopped when either OD₅₈₅>1.5 or the culture grewfor 48 hours. 240 L, defined in TABLE 15, was prepared. 100 L sugar feedwas added to a 3500 L fermenter 2004. 300 L culture in 2002 was used toinoculate in 3500 L media (8-10% v/v inoculation rate) in 2004. Another100 L of the sugar feed was added once glucose concentrations droppedbelow 2 g/L. The cultured was stopped when either OD₅₈₅>2.5 or theculture grew for 72 hours. The entire culture in 2004 was thencentrifuged under anaerobic atmosphere and harvested as a biomass. 100 Lfilter-sterilized degassed cryoprotectant, defined in TABLE 17, wasmixed with the biomass in a mixing tank purged with anaerobic gas. Thebiomass with cryoprotectant was lyophilized (frozen and dried) andground. For each step, the media used for the bacterial culture weresterilized (autoclaving at 121° C.) and degassed with N₂H₂CO₂ (90:5:5)before use. Sugar components (N-acetylglucosamine and glucose) wereprepared separately from the remaining NAGT media components. Sugar feedwas filter sterilized, degassed, and added to the remaining NAGT mediacomponents to generate the complete culture media.

FIG. 21 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 1 L A. muciniphila (DSM 33213)culture in NAGT media in a 1 L bottle in a 3500 L manufacturing process.

FIG. 22 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 20 L A. muciniphila (DSM 33213)culture in NAGT media in a 20 L fermenter in a 3500 L manufacturingprocess.

FIG. 23 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 300 L A. muciniphila (DSM33213) culture in NAGT media in a 300 L fermenter in a 3500 Lmanufacturing process.

FIG. 24 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 3500 L A. muciniphila (DSM33213) culture in NAGT media in a 3500 L fermenter in a 3500 Lmanufacturing process.

FIG. 25 shows a schematic flow chart for an optimized andultra-large-scale growth and manufacturing process for F. prausnitzii(DSM 33185) in 3500 L culture volume. 6.4 mL of Working Cell Bank (WCB)F. prausnitzii (DSM 33185) was thawed in the anaerobic chamber andinoculated in 1 L of reduced NAGT media (2% v/v inoculation rate) in a 2L flask 2501 in an anaerobic chamber. The cultured was stopped wheneither OD₆₀₀>3 or the culture grew for 48 hours. 1.5 L culture in 2501was used to inoculate in 150 L media (1% v/v inoculation rate) in a 300L fermenter 2502. The cultured was stopped when either OD₅₈₅>5 or theculture grew for 48 hours. 250 L, defined in TABLE 22, was prepared. 100L sugar feed was added to a 3500 L fermenter 2503. 35 L culture in 2502was used to inoculate in 300 L media (5% v/v inoculation rate) in a 3500L fermenter 2503. The cultured was stopped when either OD₅₈₅>5 or theculture grew for 72 hours. The entire culture in 2503 was thencentrifuged under anaerobic atmosphere and harvested as a biomass. 120 Lfilter-sterilized degassed cryoprotectant, defined in TABLE 24, wasmixed with the biomass in a mixing tank purged with anaerobic gas. Thebiomass with cryoprotectant was lyophilized (frozen and dried) andground. For each step, the media used for the bacterial culture weresterilized (autoclaving at 121° C.) and degassed with N₂H₂CO₂ (90:5:5)before use. Sugar components (glucose) was prepared separately from theremaining YFAP media components. Sugar feed was filter sterilized,degassed, and added to the remaining YFAP media components to generatethe complete culture media.

FIG. 26 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 2 L F. prausnitzii (DSM 33185)culture in YFAP media in a 2 L flask in a 3500 L manufacturing process.

FIG. 27 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 150 L F. prausnitzii (DSM33185) culture in YFAP media in a 300 L fermenter in a 3500 Lmanufacturing process.

FIG. 28 shows a chart of light absorption at 585 nanometer wavelength(Y-axis) versus time for the growth of a 3500 L F. prausnitzii (DSM33185) culture in YFAP media in a 3500 L fermenter in a 3500 Lmanufacturing process.

FIG. 29 shows the three stages (screening period, treatment period, andwashout period) of a human clinical study as described herein, as wellas its ability to treat allergy in subjects of varying age compared toplacebo, by orally administering the pharmaceutical compositions to thesubjects.

FIG. 30 shows the drug product in a solid dosage form (3001) or liquiddosage form (3002). The compositions in either form can be administeredorally.

FIG. 31 shows the three stages (screening period, treatment period, andwashout period) of a human clinical study as described herein, as wellas its ability to treat allergy in subjects of varying age compared toplacebo, by orally administering the pharmaceutical compositions to thesubjects.

FIG. 32 shows the three stages (screening period, treatment period, andwashout period) of a human clinical study as described herein, as wellas its ability to treat allergy in subjects of varying age compared toplacebo, by orally administering the pharmaceutical compositions to thesubjects.

DETAILED DESCRIPTION

Provided herein are pharmaceutical compositions comprising bacteria fortreatment of an inflammatory or metabolic disease. In some instances,the pharmaceutical compositions further comprise one or morepharmaceutically acceptable excipients. Pharmaceutical compositionsdescribed may comprise one or more bacterial species, including one ormore bacteria strains. In some instances, pharmaceutical compositionscan comprise, consist essentially of, or consist of any one or more ofLactobacillus species (sp.), Akkermansia sp., and/or Faecalibacteriumsp. In further instances, pharmaceutical compositions described hereincomprise, consist essentially of, or consist of any one or moreparticular strains of the following species referenced herein:Lactobacillus sp., Akkermansia sp., and/or Faecalibacterium sp. Forexample, in some instances, pharmaceutical compositions described hereincomprise, consist essentially of, or consists of the bacterial strainsLactobacillus crispatus(DSM 33187) (also referred to herein as “L.crispatus (DSM 33187)”), Akkermansia muciniphila with deposit ID numberDSM 33213 (also referred to herein as “A. muciniphila (DSM 33213)”), andFaecalibacterium prausnitzii (DSM 33185) (also referred to herein as “F.prausnitzii (DSM 33185)”). In some cases, pharmaceutical compositionscan comprise Composition A, as defined in Example 2.

Provided herein are pharmaceutical composition, formulations of suchcompositions, methods of manufacturing such compositions, administeringroutes for such pharmaceutical compositions, as well as indications thatmay be prevented and/or treated using such pharmaceutical compositions.

The present disclosure also provides methods of formulating apharmaceutical composition described herein, as well as methods foradministering such pharmaceutical compositions to a subject having orsuspected of having a disease or condition. Such methods can compriseformulating a pharmaceutical composition herein that can comprise orconsist of one or more bacterial species and/or strains into an oraldosage form. Such oral formulation can comprise or consist of one ormore bacterial species and/or strains, a buffered glycerol solution,e.g., one that can be composed of standard phosphate buffered salinecontaining 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄,20% v/v glycerol, and an antioxidant such as 0.1% w/w L-cysteine.

Further provided herein are methods of manufacturing that allow forproduction of batches of bacterial strain described herein. Such methodscan provide one or more advantages compared to conventional methods ofmanufacturing. Such advantages can include any one or more of (i)increased total yields, (ii) increased growth rates, and/or (iii) highernumbers of viable bacterial cells per number of total cells. In someinstances, such methods can include use of non-animal derived mediacomponents. Such non-animal media can include vegetal media. Suchvegetal media can comprise various components such as vegetal peptone,vegetal extracts, yeast extract, and other non-animal components. Insome instances, such non-animal media can include N-acetylglucosamine-threonine (NAGT) media and Boullion MRS vegetal media, Yeastfatty acid Phytone (YFAP) media, as well as modified versions thereof.Such modified media can have one or more media components removed,added, and/or substituted by other components. In other instances, theamount of a media component is increased or decreased compared to anunmodified media. In an example, a modified NAGT media may not compriseany one or more of magnesium, calcium, or glucose.

Such oral formulations of the present disclosure can be used in a methodof preventing and/or treating a disease or condition in a subject, themethod comprising administering the oral formulation to a subject (e.g.,a human) having or suspected of having the disease or condition. Suchdisease or condition can be an inflammatory disease (e.g., an allergy orasthma) or an autoimmune disease.

Definitions

Whenever the term “at least,” “greater than,” or “greater than or equalto” precedes the first numerical value in a series of two or morenumerical values, the term “at least,” “greater than” or “greater thanor equal to” applies to each of the numerical values in that series ofnumerical values. For example, greater than or equal to 1, 2, or 3 isequivalent to greater than or equal to 1, greater than or equal to 2, orgreater than or equal to 3.

Whenever the term “no more than,” “less than,” or “less than or equalto” precedes the first numerical value in a series of two or morenumerical values, the term “no more than,” “less than,” or “less than orequal to” applies to each of the numerical values in that series ofnumerical values. For example, less than or equal to 3, 2, or 1 isequivalent to less than or equal to 3, less than or equal to 2, or lessthan or equal to 1.

The term “about,” as used herein in the context of a numerical value orrange, generally refers to ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%of the numerical value or range recited or claimed, unless otherwisespecified.

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions that cancomprise, consist essentially of, or consist of a bacterial consortiumand one or more pharmaceutical excipients. Such pharmaceuticalexcipients can include a cryoprotectant, an antioxidant, and an aqueousbuffer solution.

Provided herein are pharmaceutical composition that can comprise abacterial consortium. Such bacterial consortium can comprise one or moredifferent bacterial species and/or strains. Such bacterial speciesand/or strains can belong to one or more different bacterial phyla. Suchbacterial phyla can include Verrucomicrobia, Firmicutes, Proteobacteria,Actinobacteria, and/or Bacteroidetes, or a combination thereof.

In some instances, a bacterial consortium described herein can compriseone or more Lactobacillus sp. The one or more Lactobacillus sp. caninclude Lactobacillus johnsonii, Lactobacillus rhamnosus, Lactobacilluszeae, Lactobacillus acidipiscis, Lactobacillus acidophilus,Lactobacillus agilis, Lactobacillus aviarius, Lactobacillus brevis,Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacilluscrustorum, Lactobacillus curvatus, Lactobacillus diolivorans,Lactobacillus farraginis, Lactobacillus fermentum, Lactobacillusfuchuensis, Lactobacillus harbinensis, Lactobacillus helveticus,Lactobacillus hilgardii, Lactobacillus intestinalis, Lactobacillusjensenii, Lactobacillus kefiranofaciens, Lactobacillus kefiri,Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis, or acombination thereof. In some embodiments, the Lactobacillus sp. isLactobacillus johnsonii or Lactobacillus crispatus. In such instances, abacterial consortium herein can comprise one or more Lactobacillusjohnsonii or Lactobacillus crispatus strains. Such one or moreLactobacillus crispatus strain(s) can include Lactobacilluscrispatus(DSM 33187) (i.e., L. crispatus (DSM 33187)). In variousinstances, a bacterial consortium herein comprises Lactobacilluscrispatus(DSM 33187).

In some instances, a bacterial consortium herein can comprise one ormore Akkermansia sp. Such one or more Akkermansia sp. can includeAkkermansia muciniphila, Akkermansia glycaniphila, or a combinationthereof. In some instances, the one or more Akkermansia sp. isAkkermansia muciniphila. In such instances, a bacterial consortiumherein can comprise one or more Akkermansia muciniphila strains. Suchone or more Akkermansia muciniphila strains can include Akkermansiamuciniphila (DSM 33213). In various instances, a bacterial consortiumherein comprises Akkermansia muciniphila (DSM 33213).

In some instances, a bacterial consortium herein can comprise one ormore Faecalibacterium sp. The one or more Faecalibacterium sp. caninclude Faecalibacterium prausnitzii. In such instances, a bacterialconsortium herein can comprise one or more Faecalibacterium prausnitziistrains. Such one or more Faecalibacterium prausnitzii strains caninclude Faecalibacterium prausnitzii (DSM 33185), Faecalibacteriumprausnitzii (DSM 33191), Faecalibacterium prausnitzii (DSM 33186), orFaecalibacterium prausnitzii (DSM 33190), or a combination thereof. Invarious instances, a bacterial consortium herein comprisesFaecalibacterium prausnitzii (DSM 33185).

Further provided herein are bacterial consortia that can comprise one ormore strains of any one or more of Bacteroides sp., Blautia sp.,Bifidobacterium sp., Coprococcus sp., or Dorea sp. In such instances, abacterial consortium herein can comprise any one or more of Bacteroidesfaecis (DSM 33177), Bacteroides thetaiotaomicron (DSM 33178), Blautiaproducta (DSM 33180), Bifidobacterium longum (DSM 33179), Coprococcuscomes (DSM 33176), or Dorea longicatena (DSM 33188). Exemplary strainsfor inclusion in a bacterial consortium described herein are listed inTABLE 1.

TABLE 1 Exemplary Bacterial Strains. Bacterial Strain Deposit ID #Bacterial Strain Deposit ID # A. muciniphila DSM 33213 F. prausnitziiDSM 33191 B. longum DSM 33179 F. prausnitzii DSM 33186 B. producta DSM33180 F. prausnitzi DSM 33190 B. thetaiotaomicron DSM 33178 L. crispatusDSM 33187 C. comes DSM 33176 B. faecis DSM 33177 F. prausnitzii DSM33185 Dorea lonpicatena DSM 33188

Provided herein are bacterial consortia that can comprise, consistessentially of, or consist of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bacterial species and/or strain(s). Insome instances, such bacterial consortia can comprise at least onebacterial strain selected from TABLE 1. In some embodiments, a bacterialconsortium can consist of up to 3 different bacterial strains. In someembodiments, a bacterial consortium described herein comprises at leastone, at least two, or all three bacterial strains listed in TABLE 2. Insome instances, a bacterial consortium comprises or consists of thebacterial strains L. crispatus (DSM 33187), A. muciniphila (DSM 33213),and F. prausnitzii (DSM 33185).

TABLE 2 A Subset of Bacterial Strains. Bacterial strain Deposit ID #Lactobacillus crispatus DSM 33187 Akkermansia muciniphila DSM 33213Faecalibacterium prausnitzii DSM 33185

The present disclosure provides bacterial consortia that can comprise avarying number of colony-forming units (CFU) of each of the bacterialspecies and/or strain it contains. In some instance, such bacterialconsortium can comprise from about 10{circumflex over ( )}3 CFU to about10{circumflex over ( )}12 CFU, from about 10{circumflex over ( )}4 CFUto about 10{circumflex over ( )}12 CFU, from about 10{circumflex over( )}7 CFU to about 10{circumflex over ( )}11 CFU, from about10{circumflex over ( )}8 CFU to about 10{circumflex over ( )}10 CFU, orfrom about 10{circumflex over ( )}9 CFU to about 10{circumflex over( )}10 CFU of a bacterial species or strain. In some embodiments, suchbacterial consortium can also comprise from about 10{circumflex over( )}7 CFU to about 10{circumflex over ( )}10 CFU of a bacterial speciesor strain. In some instances, a bacterial consortium can comprise atleast about 10{circumflex over ( )}3 CFU, 5×10{circumflex over ( )}3CFU, 10{circumflex over ( )}4 CFU, 5×10{circumflex over ( )}4 CFU,10{circumflex over ( )}5 CFU, 5×10{circumflex over ( )}5 CFU,10{circumflex over ( )}6 CFU, 5×10{circumflex over ( )}6 CFU,10{circumflex over ( )}7 CFU, 5×10{circumflex over ( )}7 CFU,10{circumflex over ( )}8 CFU, 5×10{circumflex over ( )}8 CFU,10{circumflex over ( )}9 CFU, 5×10{circumflex over ( )}9 CFU,10{circumflex over ( )}10 CFU, 5×10{circumflex over ( )}10, CFU,10{circumflex over ( )}11 CFU, 5×10{circumflex over ( )}11 CFU, or10{circumflex over ( )}12 CFU, but no more than about 5×10{circumflexover ( )}12 CFU of a bacterial species or strain. The bacterialconsortia can also comprise from about 10{circumflex over ( )}6 to about10{circumflex over ( )}11 CFU per bacterial species or strain. In somecases, the bacterial consortia can comprise from about 10{circumflexover ( )}3 to about 10{circumflex over ( )}12 CFU per bacterial speciesor strain. In some instances, the bacterial consortia can comprise fromabout 10{circumflex over ( )}8 to about 5×10{circumflex over ( )}10 CFUper bacterial species or strain. In some instances, the bacterialconsortia can comprise from about 10{circumflex over ( )}7 to about5×10{circumflex over ( )}10 CFU per bacterial species or strain. Invarious embodiments, a bacterial consortium can comprise about5×10{circumflex over ( )}8 CFU per bacterial species or strain. Ininstances where a pharmaceutical composition is formulated into a unitdose for administration, such CFU values can be per mass unit (e.g.,5×10{circumflex over ( )}8 CFU/g) or volume unit (e.g., 5×10{circumflexover ( )}8 CFU/mL) of such dosage form.

In some embodiments, the present disclosure provides bacterial consortiathat can comprise varying amounts of colony-forming units (CFU) ofbacterial cells. Such bacterial consortia can comprise from about10{circumflex over ( )}3 CFU to about 10{circumflex over ( )}12 CFU,from about 10{circumflex over ( )}4 CFU to about 10{circumflex over( )}12 CFU, from about 10{circumflex over ( )}7 CFU to about10{circumflex over ( )}11 CFU, from about 10{circumflex over ( )}8 CFUto about 10{circumflex over ( )}10 CFU, or from about 10{circumflex over( )}9 CFU to about 10{circumflex over ( )}11 CFU of bacterial cells.Such bacterial consortium can also comprise from about 10{circumflexover ( )}3 CFU to about 10{circumflex over ( )}12 CFU of bacterialcells. In some embodiments, such bacterial consortium can also comprisefrom about 10{circumflex over ( )}7 CFU to about 10{circumflex over( )}10 CFU of bacterial cells. In some instances, a bacterial consortiumcan comprise at least about 10{circumflex over ( )}3 CFU,5×10{circumflex over ( )}3 CFU, 10{circumflex over ( )}4 CFU,5×10{circumflex over ( )}4 CFU, 10{circumflex over ( )}5 CFU,5×10{circumflex over ( )}5 CFU, 10{circumflex over ( )}6 CFU,5×10{circumflex over ( )}6 CFU, 10{circumflex over ( )}7 CFU,5×10{circumflex over ( )}7 CFU, 10{circumflex over ( )}8 CFU,5×10{circumflex over ( )}8 CFU, 10{circumflex over ( )}9 CFU,5×10{circumflex over ( )}9 CFU, 10{circumflex over ( )}10 CFU,5×10{circumflex over ( )}10, CFU, 10{circumflex over ( )}11 CFU,5×10{circumflex over ( )}11 CFU, or 10{circumflex over ( )}12 CFU, butno more than about 5×10{circumflex over ( )}12 CFU of bacterial cells.

In some embodiments, the present disclosure provides a bacterialpopulation can be present in a total amount of about 10{circumflex over( )}3 CFU to about 10{circumflex over ( )}12 CFU of bacterial cells. Insome embodiments, a bacterial population can be present in a totalamount of at least about 10{circumflex over ( )}3 CFU, 5×10{circumflexover ( )}3 CFU, 10{circumflex over ( )}4 CFU, 5×10{circumflex over ( )}4CFU, 10{circumflex over ( )}5 CFU, 5×10{circumflex over ( )}5 CFU,10{circumflex over ( )}6 CFU, 5×10{circumflex over ( )}6 CFU,10{circumflex over ( )}7 CFU, 5×10{circumflex over ( )}7 CFU,10{circumflex over ( )}8 CFU, 5×10{circumflex over ( )}8 CFU,10{circumflex over ( )}9 CFU, 5×10{circumflex over ( )}9 CFU,10{circumflex over ( )}10 CFU, 5×10{circumflex over ( )}10, CFU,10{circumflex over ( )}11 CFU, 5×10{circumflex over ( )}11 CFU, or10{circumflex over ( )}12 CFU, but no more than about 5×10{circumflexover ( )}12 CFU of bacterial cells. In other cases, a bacterialpopulation can be present in a total amount of about 10{circumflex over( )}7 CFU to about 10{circumflex over ( )}10 CFU of bacterial cells. Insome instances, a bacterial population can be present in a total amountof about 1.5×10{circumflex over ( )}9 CFU of bacterial cells.

In some instances, the number of CFU of a bacterial species or strain ina pharmaceutical composition described herein can be a certain fractionof the number of CFU of that bacterial species or strain present in amicrobiota of a subject. The microbiota can be a gut or a vaginalmicrobiota. Such subject can be a human subject. Thus, in someinstances, the ratio of CFU of the bacterium in a pharmaceuticalcomposition to the number of CFU of such bacterium in a microbiota canbe from about 1:10{circumflex over ( )}4 to about 1:10, from about1:10{circumflex over ( )}3 to about 1:10, from about 1:10{circumflexover ( )}2 to about 1:10, or from about 1:10 to about 5:1. In certainembodiments of this disclosure, such ratio can be at least about 0.0001,0.0002, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1,1, 2, 3, 3.5, 4, or 5, but no more than 10.

In some embodiments herein, a bacterial consortium for use in apharmaceutical composition of this disclosure can comprise or consist ofabout 5×10{circumflex over ( )}8 CFU/mL of any of the bacterial strainsL. crispatus (DSM 33187), A. muciniphila (DSM 33213), and/or F.prausnitzii (DSM 33185). In such instances, the bacterial consortium canconsist of about 5×10{circumflex over ( )}8 CFU/mL of the bacterialstrains L. crispatus (DSM 33187), A. muciniphila (DSM 33213), and F.prausnitzii (DSM 33185).

Provided herein are pharmaceutical compositions that can comprise one ormore cryoprotectant. Such cryoprotectant can be used to maintainviability of the bacterial cells in a pharmaceutical composition whensuch composition is frozen or lyophilized, for example, during transportand/or storage prior to use. In some instances, the one or morecryoprotectant can be glycerol, dimethylsulfoxide (DMSO), ethyleneglycol, propylene glycol, 2-methyl-2,4-pentanediol, trehalose, sucrose,diethyl glycol, triethylene glycol, polyvinylpyrrolidone (PVP),polyethylene glycol (PEG), saccharose, formamide, glycerol 3-phosphate,proline, methyl alcohol, glucose, bovine serum albumin, polyvinylalcohol, hydroxyethyl starch, sorbitol, or a combination thereof.Cryoprotectant can comprise an ice blocker. An ice blocker can comprisepolyglycerol, polyvinyl alcohol, X-1000 and Z-1000. Such cryoprotectantcan be used in a pharmaceutical composition in an amount of about 5, 10,15, 20, 25, or 30 volume percent (% v/v) or weight percent (% w/w),e.g., depending on whether the pharmaceutical composition is a soliddosage from (e.g., a capsule or tablet) or a liquid dosage from (e.g., asuspension or a gel). Cryoprotectant can also comprise a carbohydrate oran antioxidant. A carbohydrate can comprise trehalose, sucrose,sorbitol, glucose, fructose, saccharose, or a combination thereof.

In some embodiments, pharmaceutical compositions described hereinfurther comprise an antioxidant. In some embodiments, the antioxidant isL-cysteine. In some embodiments, the L-cysteine is present, by weight,in an amount of about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%,1.5%, 2%, 5%, 10%, 0.001% to 0.005%, 0.0051% to 0.01%, 0.011% to 0.05%,0.05% to 0.1%, 0.051% to 0.1%, 0.11% to 0.5%, 0.51% to 1%, 1.1% to 1.5%,1.5% to 2%, 2.1% to 5%, or 5.1% to 10%. Saccharose can be present, byweight, in an amount of about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 0.1% to 1%, 1% to 5%, 5% to 10%, 10to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35%, 35 to 40%, 40 to45%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%,75 to 80%, 51 to 61%, 52 to 62%, 53 to 63%, 54 to 64%, 55 to 65%, 56 to66%, 57 to 67%, 58 to 68%, or 59 to 69%. Trehalose can be present, byweight, in an amount of about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%,2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%,9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%,15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%,21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 0.01% to 15%, 0.1% to20%, 0.01% to 0.1%, 0.11% to 1%, 1 to 11%, 2 to 12%, 3 to 13%, 4 to 14%,5 to 15%, 6 to 16%, 7 to 17%, 8 to 18%, 9 to 19%, 10 to 20%, 11 to 21%,12 to 22%, 13 to 23%, 14 to 24%, or 15 to 25%. Glycerol can be present,by volume, in an amount of about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 1 to 21%, 2 to 22%, 3 to 23%, 4 to 24%, 5 to 25%, 6to 26%, 7 to 27%, 8 to 28%, 9 to 29%, 10 to 30%, 11 to 31%, 12 to 32%,13 to 33%, 14 to 34%, 15 to 35%, 16 to 36%, 17 to 37%, 18 to 38%, 19 to39%, or 20 to 40%.

In some embodiments herein, the cryoprotectant of a pharmaceuticalcomposition herein is glycerol. Such glycerol can be used in an amountof about 20% v/v in a pharmaceutical composition that can comprise abacterial consortium of one or more, two or more, or three or morebacterial strains selected from TABLE 1. In some embodiments, thebacteria populations can be lyophilized. A lyophilization process cancomprise a low temperature dehydration of the bacterial population. Insome embodiments, the lyophilization process can comprise subjecting thebacterial population at low temperature and low pressure.

Provided herein are pharmaceutical compositions that can comprise one ormore antioxidant. In some instances, such antioxidant can be used toprotect anaerobic bacterial species and/or strain(s) that may be presentin the pharmaceutical composition. In such instances, the one or moreantioxidant can be used to provide anaerobic conditions during storageand/or transport, and/or to protect the bacterial cells from reactiveoxygen species. In some embodiments herein, the antioxidant can beascorbic acid, dithiothreitol, glutathione, phenolic acids (e.g.,gallic, protocatechuic, caffeic, and rosmarinic acids), phenolicditerpenes (e.g., carnosol and carnosic acid), flavonoids (e.g.,quercetin and catechin), volatile oils (e.g., eugenol, carvacrol,thymol, and menthol), α-Tocopherol (e.g., vitamin E), Trolox, ascorbicacid, vitamin A, vitamin C, coenzyme Q10, manganese, iodide, melatonin,alpha-carotene, astaxanthin, beta-carotene, canthaxanthin,cryptoxanthin, lutein, lycopene, zeaxanthin, flavonoids (e.g., flavonessuch as apigenin), luteolin, tangeithin, flavonols, isorhamnetin,kaemferol, myricetin, proanthocyanidins, quercetin, eriodictyol,hesperetin, naringenin, catechin, gallocatechin, epicatechin,epigallocatechin, theaflavine, thearubigins, isoflavone phytoestrogens,daidzein, genistein, glycitein, stilbenoids such as resveratrol,pterostilbene, anthocyanins, cyanidin, delphinidin, malvidin,pelargonidin, peonidin, petunidin, chicoric acid, chlorogenic acid,cinnamic acid, ellagic acid, ellagitannins, gallic acid, gallotannins,rosmarinic acid, curcumin, xanthones, capsaicin, bilirubin, citric acid,oxalic acid, phytic acid, N-acetylcysteine, L-cysteine, L-glutamate,L-proline, R-α-lipoic acid, anthocyanins, copper, cryptoxanthins,flavonoids, indoles, isoflavonoids, lignans, selenium, zinc, or acombination thereof. Such one or more antioxidant(s) can be present in apharmaceutical composition in an amount of about 0.05%, 0.1%, 0.2%,0.3%, 0.4%, or 0.5% w/w. L-glutamate can be present, by weight, in anamount of about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.1%, 2.2%,2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%,3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%,4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%,5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 8%,9%, 10%, 1 to 5%, 1.1 to 5.1%, 1.2 to 5.2%, 1.3 to 5.3%, 1.4 to 5.4%,1.5 to 5.5%, 1.6 to 5.6%, 1.7 to 5.7%, 1.8 to 5.8%, 1.9 to 5.9%, 2 to6%, 2.1 to 6.1%, 2.2 to 6.2%, 2.3 to 6.3%, 2.4 to 6.4%, 2.5 to 6.5%, 2.6to 6.6%, 2.7 to 6.7%, 2.8 to 6.8%, 2.9 to 6.9%, 3 to 7%, 3.1 to 7.1%,3.2 to 7.2%, 3.3 to 7.3%, 3.4 to 7.4%, 3.5 to 7.5%, 3.6 to 7.6%, 3.7 to7.7%, 3.8 to 7.8%, 3.9 to 7.9%, or 4 to 8%. In some case, thecryoprotectant can comprise, by weight, about 60% saccharose, about 10%trehalose, about 1% L-cysteine, and about 4% L-glutamate.

Provided herein are pharmaceutical compositions that can comprise anaqueous buffer solution. Such aqueous medium can be used as the mainstorage and transport medium for the bacterial cells. As such, thebuffer can contain any one or more of bacterial consortium,cryoprotectant, and antioxidant, either dissolved or suspended, to forma pharmaceutical composition as described herein. In some instances, theaqueous buffer solution can be phosphate buffered saline (PBS), HEPES,or Tris buffer, any other suitable buffer, or any combination thereof.In some embodiments, the buffer is PBS and comprises 137 mM NaCl, 2.7 mMKCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄. In other cases, the buffer can bePBS and can have a pH of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2,8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.

Thus, in some embodiments herein, a pharmaceutical composition comprisesa bacterial consortium consisting of about 5×10{circumflex over ( )}8CFU of each of the bacterial strains A. muciniphila (DSM 33213), F.prausnitzii (DSM 33185), and L. crispatus (DSM 33187), about 20% v/vglycerol as cryoprotectant, 0.1% w/w L-cysteine as antioxidant, and PBSbuffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mMKH₂PO₄. Such pharmaceutical composition can be manufactured andformulated into an orally administrable dosage form using the methodsand compositions described herein.

Provided herein are pharmaceutical compositions that can be formulatedfor administration to a subject. The subject can be a human subject.Administration can include parenteral administration and oraladministration. Parenteral administration can include administering apharmaceutical composition in various non-oral routes, e.g., in the formof a suppository. In various other instances, a pharmaceuticalcomposition described herein can be formulated into an oral dosage from.Such oral dosage form can include a capsule, tablet, emulsion,suspension, syrup, gel, gum, paste, herbal tea, drops, dissolvinggranules, powders, tablets, lyophilizate, and any other suitable oraldosage forms. A capsule can comprise a plant-derived material. Theplant-derived material can comprise a cellulose-based polymer. A capsulecan also comprise gelatin; hydroxypropyl methylcellulose (HPMC); starch;hydrolyzed collagen (acid, alkaline, enzymatic, or thermal hydrolysis)from animal origin or cellulose-based; pullulan; tapioca; or anycombination thereof. A cellulose-based polymer can comprise pullulan. Acapsule can be enteric-coated. An enteric-coated capsule can comprisefatty acids, waxes, shellac, plastics, plant fibers, or any combinationthereof. A capsule can have a size of 000, 00, 0, 1, 2, 3, 4, or 5 EmptyPill Capsule Size. A capsule can be starch-free, gluten-free, andpreservative-free. >90% of a capsule dissolves in water, pH=1.2solution, sodium acetate buffer USP (pH=4.5), or sodium phosphate buffer(pH=7.2) within 60 minutes, when measured by the dissolution ofacetaminophen when the capsule is filled with unformulatedacetaminophen. A capsule can have a disintegration endpoint of about 1.6minutes, as measured at 37° C. with de-ionized water. A capsule can havea disintegration endpoint of about 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 3.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4minutes, as measured at 37° C. with de-ionized water. A capsule can havea disintegration endpoint of 0.1 to 0.5 minutes, 0.51 to 0.6 minutes,0.61 to 0.7 minutes, 0.71 to 0.8 minutes, 0.81 to 0.9 minutes, 0.91 to 1minutes, 1.01 to 1.1 minutes, 1.11 to 1.2 minutes, 1.21 to 1.3 minutes,1.31 to 1.4 minutes, 1.41 to 1.5 minutes, 1.51 to 1.6 minutes, 1.61 to1.7 minutes, 1.71 to 1.8 minutes, 1.81 to 1.9 minutes, 1.91 to 2minutes, 2.01 to 2.1 minutes, 2.11 to 2.2 minutes, 2.21 to 2.3 minutes,2.31 to 2.4 minutes, 2.41 to 2.5 minutes, 2.51 to 2.6 minutes, 2.61 to2.7 minutes, 2.71 to 2.8 minutes, 2.81 to 2.9 minutes, 2.91 to 3minutes, 3.01 to 3.1 minutes, 3.11 to 3.2 minutes, 3.21 to 3.3 minutes,3.31 to 3.4 minutes, 3.41 to 3.5 minutes, 3.51 to 3.6 minutes, 3.61 to3.7 minutes, 3.71 to 3.8 minutes, 3.81 to 3.9 minutes, or 3.91 to 4minutes, as measured at 37° C. with de-ionized water. A capsule can havean oxygen permeability (cm³/m²/day) of ≤0.5, as measured by a gascomposition in the capsule. A capsule can have an oxygen permeability(cm³/m²/day) of ≤0.0001, ≤0.0005, ≤0.001, ≤0.005, ≤0.01, ≤0.05, ≤0.1,≤0.5, ≤1, ≤1.5, ≤2, ≤5, or ≤10, as measured by a gas composition in thecapsule.

Further provided herein are oral formulations of the pharmaceuticalcompositions that can be frozen. Such frozen formulations can beadministered in a frozen state to a subject, such as a human subject. Insome instances, such frozen formulation can be a popsicle, an ice cream,or other frozen formulations.

In various embodiments herein, a pharmaceutical composition of thisdisclosure can be in a liquid suspension for oral administration to asubject. Such liquid suspension can be aliquoted into certain volumes toprovide a unit dose of such oral dosage form. Such unit dose can have avolume of about 0.25, 0.5, 1, 2, 3, 5, or 10 mL. In some instances, theunit dose of a pharmaceutical composition herein has a volume of about 1mL. Such pharmaceutical composition can comprise a bacterial consortium,a cryoprotectant, an antioxidant, an aqueous buffer solution that canfrom a liquid cell suspension. Such cell suspension can be tested forquality control to ensure it contains a certain number of metabolicallyactive cells per bacterial strain as described herein.

In some embodiments, provided herein are pharmaceutical compositionsformulated into a unit dose for oral administration to a subject. Suchoral formulation can comprise or consist of about 5×10{circumflex over( )}8 CFU of each of the bacterial strains A. muciniphila (DSM 33213),F. prausnitzii (DSM 33185), and L. crispatus (DSM 33187), about 20% v/vglycerol, about 0.1% w/w L-cysteine, and PBS buffer containing 137 mMNaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄. Such oralformulation can have a total volume of about 1 mL.

Provided herein are methods for manufacturing the pharmaceuticalcompositions described herein. In some instances, such pharmaceuticalcompositions comprise a bacterial consortium comprising one or morebacterial species and/or strains. In some instances, such one or morebacterial strains can include any one or more of L. crispatus (DSM33187), A. muciniphila (DSM 33213), and/or F. prausnitzii (DSM 33185).Manufacturing of such pharmaceutical compositions can comprise severalsteps. Such steps can include growth media preparation, inoculation andculture, harvest of the bacterial cells, and assembly of a bacterialconsortium by combining the prepared bacterial strain batches for use ina pharmaceutical composition. In some instances, such manufacturingmethods can be used for preparation of a pharmaceutical composition forclinical use in human subjects.

Methods of this disclosure for manufacturing bacterial consortia caninclude media preparation, which can involve dissolving various drymedia components such as salts, vitamins, antioxidants, etc., in USPgrade water for injection. After complete dissolution, the pH of themedia can be adjusted to ensure optimal growth of the respectivebacterial cells. The pH adjusted media can then be transferred to abiosafety cabinet and sterilized. In various instances, a microbialconsortium herein can comprise one or more anaerobic bacterial strains.In such instances, media can be transferred to an anaerobic chambercontaining an atmosphere of about N₂H₂CO₂ (90:5:5) to reduce prior toinoculation of anaerobic bacteria.

The manufacturing methods herein can comprise generating a starterculture of the bacterial strains/species. Such methods can includegenerating a starter culture for the bacterial strains to be included ina pharmaceutical composition by using a certain volume from each flaskcontaining filtered media and transfer such volume to a sterile,pre-reduced screw cap tube, followed by a transfer of thawed bacterialcells using a stock solution from a cell bank that contains therespective bacterial cells. In instances where anaerobic bacterial cellsare used, the starter cultures can be grown at about 37° C. for about12-16 hours under anaerobic conditions. After a certain incubation timeperiod, the starter cultures can be visibly inspected for growth(turbidity) and, upon confirmation of bacterial growth, transferred intoadditional, larger culture flasks for cell expansion. After incubationfor about 12, 18, 24, 30 hours, the cell density and absorbance of thecell culture medium can be measured. Such measurements can be performedby measuring the absorbance at of the cell suspension at 600 nanometersto ensure the absorbance falls within a specified OD₆₀₀ range. SuchOD₆₀₀ value or range can be specific a bacterial strain. For example,each bacterial strain shown in TABLE 1 can have a specific OD₆₀₀ valueor range. Such OD₆₀₀ range can be from about 0.5 to about 1.5, fromabout 0.7 to about 1.3, or from about 0.9 to about 1.1. Subsequent toOD₆₀₀ measurements, the bacterial cells can be harvested using varioustechniques such as centrifugation.

In some instances, for cell harvest, appropriate centrifugationparameters can be selected. For example, in some instances, any of thebacterial strains listed in TABLE 1 can be harvested using parametersthat include rotation at about 5,000-10,000×g for about 20-60 mins atabout 4° C. to ensure the cells can be separated from a supernatant.Following centrifugation, the resulting cell pellets can be transferredback into an anaerobic environment (e.g., an anaerobic chamber) and theclarified culture supernatant can be completely removed from suchpellets using, e.g., a sterile serological pipette. The cell pellets canthen be combined by resuspension in a concentrated and pre-reducedcryoprotectant solution. In some instances, such suspension can be about20-40 times as concentrated as the cell suspension during growth and thepre-reduced cryoprotectant solution can comprise about 20% v/v glycerolor another cryoprotectant. The concentrated cell suspensions can then bedispensed into aliquots, e.g., into pre-reduced, pre-labeled 2 mL screwcap cryovials and transferred to pre-labeled storage boxes at about −70°C. or lower. In instances where such bacterial cells may be used in apharmaceutical composition, specification testing can be conducted aboutthree days after manufacturing and initial storage at about −70° C. orlower.

Provided herein are methods for manufacturing cell populations or cellbatches of one or more species and/or strain(s) for their use in apharmaceutical composition. In various instances, any of the bacterialstrains shown in TABLE 1 can be used in the manufacturing methodsdescribed herein. In certain instances, one or more of the strains L.crispatus (DSM 33187), A. muciniphila (DSM 33213), and/or F. prausnitzii(DSM 33185) (TABLE 2) can be used to manufacture cell batches for use ina pharmaceutical composition. In such instances, the present disclosureprovided methods for manufacturing such cell batches.

Provided herein are methods for manufacturing L. crispatus (DSM 33187)cell batches that can be used in a pharmaceutical composition describedherein. Such methods can comprise preparing a L. crispatus (DSM 33187)cell culture medium. Such culture medium can be a vMRS medium orBoullion vMRS broth. In some instances, such culture medium is notHiMedia vMRS broth. Such culture medium can be specific for a L.crispatus (DSM 33187) strain and can comprise vMRS powder anddipotassium phosphate (K₂HPO₄). In such instances, a medium for growingand culturing L. crispatus (DSM 33187) cells can comprise about 250-300g of vMRS powder and dipotassium phosphate (K₂HPO₄). In certaininstances, such medium can comprise about 273 g vMRS powder and about12.5 g dipotassium phosphate (K₂HPO₄) and about 4.9 L of water. The pHof such vMRS media can be adjusted to about 6.5±0.1 using, e.g., 5 Mhydrochloride solution or glacial acetic acid. The media can then befiltered, reduced to an anaerobic state, and transferred to, e.g., astarter culture tube containing stock L. crispatus (DSM 33187) solution,and incubated at 37° C. for about 16-20 hours. Following incubation andexpansion, the absorbance of the cell culture at 600 nm can bedetermined and repeated in triplicates to ensure absorbance of the cellsuspension falls within the range from about 0.8 to about 1.6,preferably of about 1.0-1.4. The contents of the culture flasks can becentrifuged, the residual cell pellets re-suspended in 25 mL of sterilePBS containing an antioxidant and cryoprotectant such as 20% v/vglycerol, and then combined to yield a homogenous cell suspension. TheL. crispatus (DSM 33187) cell suspension can be aliquoted, e.g., intocryovials, to achieve a final cell concentration. Such final L.crispatus (DSM 33187) cell concentration can be from about5×10{circumflex over ( )}8 to about 10{circumflex over ( )}10 live L.crispatus (DSM 33187) cells per unit dose. Such unit dose can have avolume of about 1 mL. In such instances, the unit dose can compriseabout 5×10{circumflex over ( )}8 live L. crispatus (DSM 33187) cells.

Further provided herein are methods for manufacturing A. muciniphila(DSM 33213) cell batches that can be used in a pharmaceuticalcomposition described herein. Such methods can comprise preparing an A.muciniphila (DSM 33213) cell culture medium. In some instances, such A.muciniphila (DSM 33213) culture medium can be a modified NAGT medium.Such modified NAGT medium can contain soytone or N-acetyl glucosamine(NAG), or both soytone and NAG. In some cases, such modified NAGT mediummay not contain magnesium, calcium, glucose, or a combination thereof.In some instances, a modified NAGT medium can provide improved cellgrowth. Such improved cell growth can be about 30%, 35%, 40%, 45%, or50% higher compared to cell growth in unmodified NAGT medium.

Thus, in some instance, such NAGT culture medium can be specific for aA. muciniphila strain (DSM 33213) and can comprise any one or more ofthe ingredients: soytone, pea peptone, yeast extract, sodium bicarbonate(NaHCO₃), dibasic potassium phosphate (K₂HPO₄), sodium chloride (NaCl),magnesium sulfate (e.g., MgSO₄×7 H₂O), calcium chloride (CaCl₂),glucose, N-acetylglucosamine, L-threonine, and/or L-cysteine. In suchinstances, a volume of about 5 L of a modified NAGT medium for growingand culturing A. muciniphila (DSM 33213) cells can comprise from about75 g to 100 g of SOLABIA Pea Peptone, from about 75 g to about 85 g ofDifco™ Select Soytone, from about 10 g to about 15 g of Bacto™ YeastExtract, from about 2 g to about 8 g of sodium bicarbonate (NaHCO₃),from about 10 g to about 15 g of dibasic potassium phosphate (K₂HPO₄),from about 0.5 g to about 5 g of sodium chloride (NaCl), from about 0.5g to about 5 g of magnesium sulfate heptahydrate (MgSO₄×7 H₂O), fromabout 0.5 g to about 5 g of calcium chloride (CaCl₂), from about 20 g toabout 25 g of glucose (dextrose), from about 25 g to about 30 g ofN-acetylglucosamine, from about 15 g to about 25 g of L-threonine,and/or from about 2 g to about 8 g of L-cysteine. In one example, avolume of about 5 L of a modified NAGT medium for growing and culturingA. muciniphila (DSM 33213) cells can comprise about 82.5 g SOLABIA PeaPeptone, 82.5 g of Difco™ Select Soytone, about 12.5 g of Bacto™ YeastExtract, about 5 g of sodium bicarbonate (NaHCO₃), about 12.5 g ofdibasic potassium phosphate (K₂HPO₄), about 1.5 g of sodium chloride(NaCl), about 0.5 g of magnesium sulfate heptahydrate (MgSO₄×7 H₂O),about 0.5 g of calcium chloride (CaCl₂), about 22.6 g of glucose(dextrose), about 27.7 g of N-acetylglucosamine, about 20 g ofL-threonine, and/or about 5 g of L-cysteine.

The pH of such NAGT media can be adjusted, e.g., to about 6.5±0.1 using,e.g., 5 M hydrochloride solution. The pH of such NAGT media can also beadjusted to about 7. A. muciniphila (DSM 33213) bacterial cells can beadded into prepared vials containing such NAGT growth medium. Followingincubation for a time period that can be specific for the A. muciniphila(DSM 33213) strain, the absorbance of the cell culture at 600 nm can bemeasured and recorded to achieve an absorbance value of about 0.5 toabout 1.2, preferably about 0.7-1.1. The contents of the culture flaskscan then be centrifuged, the supernatants removed, and the residual cellpellets re-suspended in sterile PBS containing an antioxidant andcryoprotectant such as 20% v/v glycerol. The A. muciniphila (DSM 33213)cell suspension can be aliquoted into cryovials to achieve a final A.muciniphila (DSM 33213) cell concentration from about 5×10{circumflexover ( )}8 to about 10{circumflex over ( )}10 live A. muciniphila (DSM33213) cells per unit dose. Such unit dose can have a volume of about 1mL. In such instances, the unit dose can comprise about 5×10{circumflexover ( )}8 live A. muciniphila (DSM 33213) cells.

Further provided herein are methods for manufacturing F. prausnitzii(DSM 33185) cell batches that can be used in a bacterial consortium of apharmaceutical composition described herein. Such methods can comprisepreparing a complete vitamin mix solution (e.g., YFAP vitamin mix) and aF. prausnitzii (DSM 33185) cell culture medium. The YFAP vitamin mix canbe specific for the F. prausnitzii (DSM 33185) strain and can compriseany one or more of biotin, cobalamin, p-aminobenzoic acid, folic acid,pyridoxamine, thiamine, and/or riboflavin. In such instances, a 1 Lvolume of the YFAP vitamin mix can comprise about 10 mg of biotin, about10 mg of cobalamin, about 30 mg of p-aminobenzoic acid, about 50 mg offolic acid, about 150 mg of pyridoxamine, about 50 mg of thiamine, andabout 50 mg of riboflavin. All media components can be dissolved,resulting in a solution that is clear and free of solids andprecipitates. The YFAP Vitamin mix medium can be filtered and sterilizedfor use in a F. prausnitzii (DSM 33185) culture medium as describedbelow.

Such F. prausnitzii (DSM 33185) culture medium can be prepared tocomprise any one or more of BBL™ Phytone Peptone, SOLABIA Pea Peptone,Difco™ Select Soytone, Bacto™ Yeast Extract, sodium bicarbonate(NaHCO₃), dibasic potassium phosphate (K₂HPO₄), sodium chloride (NaCl),magnesium sulfate heptahydrate (MgSO₄×7 H₂O), sodium acetate (NaOAc),glucose (dextrose), sodium propionate, L-cysteine, and/or YFAP VitaminMix solution, e.g., prepared as described above. In such instances, avolume of about 5 L of the F. prausnitzii (DSM 33185) culture medium cancomprise from about 75 g to 100 g of SOLABIA Pea Peptone, from about 45g to about 55 g of BBL™ Phytone Peptone, from about 45 g to about 55 gof Difco™ Select Soytone, from about 20 g to about 30 g of Bacto™ YeastExtract, from about 2 g to about 8 g of sodium bicarbonate (NaHCO₃),from about 10 g to about 15 g of dibasic potassium phosphate (K₂HPO₄),from about 2 g to about 8 g of sodium chloride, from about 0.5 g toabout 2 g of magnesium sulfate heptahydrate (MgSO₄×7 H₂O), from about 20g to about 30 g of sodium acetate (NaOAc), from about 40 g to about 60 gof glucose (dextrose), from about 2 g to about 8 g of sodium propionate,from about 2 g to about 8 g of L-cysteine, and about 0.5 to about 3 mLof YFAP Vitamin Mix solution, e.g., prepared as described above. Thus,in an example, a volume of about 5 L of the F. prausnitzii (DSM 33185)culture medium can comprise about 100 g SOLABIA Pea Peptone, 50 g ofBBL™ Phytone Peptone, about 50 g of Difco™ Select Soytone, about 25 g ofBacto™ Yeast Extract, about 5 g of sodium bicarbonate (NaHCO₃), about12.5 g of dibasic potassium phosphate (K₂HPO₄), about 5 g of sodiumchloride, about 1 g of magnesium sulfate heptahydrate (MgSO₄×7 H₂O),about 25 g of sodium acetate (NaOAc), about 50 g of glucose (dextrose),about 5 g of sodium propionate, about 5 g of L-cysteine, and about 1 mLof YFAP Vitamin Mix solution, e.g., prepared as described above.

A F. prausnitzii (DSM 33185) culture medium, YFAP-NU, can also beprepared to comprise any one or more of Pea Peptone, NuCel® 783 YeastExtract, sodium bicarbonate (NaHCO₃), dibasic potassium phosphate(K₂HPO₄), sodium chloride (NaCl), magnesium sulfate heptahydrate(MgSO₄×7 H₂O), sodium acetate (NaOAc), glucose (dextrose), L-cysteine,and/or cobalamin. In such instances, a volume of about 5 L of the F.prausnitzii (DSM 33185) culture medium can comprise from about 75 g to100 g pea Pea peptone, about 50 g NuCel® 783 Yeast Extract, 5 g sodiumbicarbonate (NaHCO₃), about 12.5 g dibasic potassium phosphate (K₂HPO₄),about 5 g sodium chloride (NaCl), about 1 g magnesium sulfateheptahydrate (MgSO₄×7 H₂O), about 25 g sodium acetate (NaOAc), about 50g glucose (dextrose), about 5 g L-cysteine, and about 5 g cobalamin.

The pH of such cell media can be adjusted to about 6.5±0.1, e.g., usingglacial acetic acid. Such pH may vary from about 6.2 to about 6.8,depending on the bacterial strain used. In some cases, the pH of thecell media may not be regulated. Such pH may vary from about 4.5 toabout 7.5. In some embodiment, the pH of such media can be about 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5. After reduction to ananaerobic state, starter cultures for F. prausnitzii (DSM 33185) can beprepared by adding a certain volume of stock F. prausnitzii (DSM 33185)solution, e.g., approximately 500 μL of a cell bank stock solution, tostarter culture tubes containing reduced culture medium, followed byincubation at 37° C. for about 12-16 hours. After expansion of thestarter culture (e.g., after about 12-24 additional hours ofincubation), the absorbance of the cell culture at 600 nm can bedetermined and repeated in triplicates to ensure absorbance is in aspecific range. Such absorbance range can be from about 1.2 to about2.0, preferably from about 1.4 to about 1.8. The culture flasks can thenbe centrifuged, the supernatants removed, and the residual cell pelletsre-suspended in sterile PBS to yield a homogenous solution. In someinstances, the F. prausnitzii (DSM 33185) cell suspension can bealiquoted into cryovials (e.g., 2 mL cryovials) to achieve a final F.prausnitzii (DSM 33185) cell concentration from about 5×10{circumflexover ( )}8 to about 10{circumflex over ( )}10 live F. prausnitzii (DSM33185) cells per unit dose. Such unit dose can have a volume of about 1mL. In such instances, such unit dose can comprise about 5×10{circumflexover ( )}8 live F. prausnitzii (DSM 33185) cells.

The present disclosure also provides methods comprising assembling oneor more cell batches of strains to be included in a bacterial consortiumfor used in a pharmaceutical composition. For example, in someinstances, such methods comprise assembling cell populations of one ormore bacterial strains of TABLE 1. In such instances, cell batchesmanufactured for any one or more of the strains L. crispatus (DSM33187), A. muciniphila (DSM 33213), and F. prausnitzii (DSM 33185) canbe combined to form a bacterial consortium for use in a pharmaceuticalcomposition.

Such methods can comprise determining the amount of metabolically activecells in each cell population of a bacterial strain. In instances wherethe bacterial consortium of a pharmaceutical composition comprises orconsists of the three bacterial strains L. crispatus (DSM 33187), A.muciniphila (DSM 33213), and F. prausnitzii (DSM 33185), the number ofmetabolically active bacterial cells in each of the prepared cellbatches can be determined. Such measurements can be performed using anysuitable method, e.g., those described herein. The information obtainedfrom such measurements can be used to determine the amount unit dosesthat can be prepared from a given batch of a bacterial strain (e.g., L.crispatus (DSM 33187), A. muciniphila (DSM 33213), and/or F. prausnitzii(DSM 33185)). For example, the amount of unit doses that can be preparedfrom a L. crispatus (DSM 33187) batch can be determined as follows:number of potential doses from L. crispatus (DSM 33187) batch=((L.crispatus (DSM 33187) Average Potency in CFU/mL)×(L. crispatus (DSM33187) Batch Volume in mL))/(4×10{circumflex over ( )}8 CFU/dose).

Thus, in some embodiments, a bacterial consortium of a pharmaceuticalcomposition herein can comprise about 5×10{circumflex over ( )}8 CFU perbacterial species or strain. Once the number of potential unit dosesthat can be generated from each strain has been calculated, therespective vials containing the cells of the respective strain can beremoved from the freezer and allowed to pre-reduce in an antechamberprior to proceeding. Subsequent to reduction and thawing, the calculatedamounts of cell suspension volumes for each strain can be transferred toa 1 L glass bottle and the volume increased using a buffer such as PBSto arrive at the calculated concentration of cells per milliliter. Insome cases, such calculated concentration can be about 5×10{circumflexover ( )}8 CFU/bacterial species/mL of pharmaceutical composition. Theresulting homogenous suspension can be aliquoted into unit doses usingcryovials and can be stored at −80° C. until further use such asadministration to a subject.

The manufacturing methods of a bacterial consortium provided herein canfurther comprise performing a quality control to ensure the cells of thebacterial strains in the respective composition are viable andcorrespond to the correct strain(s). In such quality control methods,various parameters, test methods and specifications can be evaluated foreach strain batch. Such evaluation can be performed prior toadministration of the pharmaceutical composition to a subject. Exemplaryquality control parameters can include (i) the concentration of thebacterial strain(s), and (ii) the morphology by visual inspection ofcolony growth. For example, the morphology of F. prausnitzii (DSM 33185)cell colonies can include a circular, entire margin, flat,small-moderate size, cream-tan color; the morphology of A. muciniphila(DSM 33213) cell colonies can include a circular, entire margin, raised,punctiform size, opaque-translucent for A. muciniphila (DSM 33213)cells; and the morphology of L. crispatus (DSM 33187) cell colonies caninclude a circular, entire margin, raised, small-moderate, white-creamcolor.

Provided herein are pharmaceutical compositions that can be designed andmanufactured to allow storage and/or transport of the pharmaceuticalcompositions. In some instances, a pharmaceutical composition hereincomprising a bacterial consortium can be designed such that theviability of the bacterial cells in the pharmaceutical composition isnot or only minimally affected by storage and/or transport. In suchinstances, the viability of at least about 80%, 85%, 90%, 95%, 97%, or99% of bacterial cells in the pharmaceutical composition is maintainedduring storage and/or transport.

In some instances, a pharmaceutical composition herein comprises acryoprotectant to allow storage at low temperatures at about −70° C. or−80° C. to preserve viability of the bacterial cells. In such instances,the pharmaceutical composition can comprise about 20% v/v glycerol as acryoprotectant. A pharmaceutical composition herein can further comprisean antioxidant that can preserve an anaerobic environment in the storageor transport vial and can protect the bacterial cells from reactiveoxygen species.

In one example, in a pharmaceutical composition herein, live, vegetativebacteria can be preserved frozen in phosphate buffered saline (PBS) with20% v/v glycerol and 0.1% w/w cysteine to preserve their viability. Insuch instances, the live bacteria can belong to any one or more of thestrains shown in TABLE 1.

The present disclosure provides containers and kits that can be used incombination with the herein described pharmaceutical compositions. Thepresent disclosure further provides instructions that can direct a user(e.g., a human user) to use such containers and kits that comprise thepharmaceutical composition.

In some embodiments, pharmaceutical compositions described herein arepresent in a container. The container can be used to grow, store,transport, aliquot, and/or administer a pharmaceutical composition ofthis disclosure. For example, such container can be used to administer apharmaceutical composition to a subject. In an example, the container isa cryovial and can be used to administer pharmaceutical composition to ahuman subject. A container described herein can also provide conditionssuitable for growth, transport, and/or storage (e.g., cooled or frozenstorage) of bacterial populations, e.g., those populations that compriseone or more anaerobic bacterial cells. Such anaerobic bacterial cellscan include any one or more of A. muciniphila (DSM 33213), F.prausnitzii (DSM 33185), and/or L. crispatus (DSM 33187) cells. In suchcases, a container may be used to provide a certain oxygen content orconcentration during growth, transport, and/or storage of apharmaceutical composition in order to preserve the viability of thebacterial cells. In some instances, a container herein can preserve theviability of at least about 80%, 85%, 90%, 95%, 97%, or 99% of bacterialcells in a pharmaceutical composition. In some instances, a containercan preserve the viability of about 95% of bacterial cells for at leastabout 1 week, 2 weeks, 4 weeks, 8 weeks, or 12 weeks. Containers canfurther be used to provide suitable volumes, amounts, and dosingschedules for administration of such pharmaceutical composition to asubject. In such instances, a container or kit comprising such containercan be designed for self-administration by a human subject. Instructionsfor such self-administration can be provided as user instructions andcan be part of a kit described herein. In various instances, suchinstructions can be written instructions or oral instructions, or acombination thereof.

In some embodiments, pharmaceutical compositions described herein arepresent in a container. The container can comprise a 2 mL polypropylenescrew cap vial. A vial can be a single dose vial or a multi-dose vial.In some cases, a container can also comprise cyclic olefin copolymer(COC), cyclic olefin polymer (COP), polypropylene, polyethylene (HDPE),ethylene-vinyl alcohol (EVOH)-based material, glass, plastic tubes,jars, aluminum tubes, dispenser tubes, or any combinations thereof. Thevolume of a vial can be 1/50, 1/10, 1/5, 1/3, 1/2, 5/8, 1, 2, 3, 4, 8,11, 13, 16, 20, 30, 40, 50 DRAM. The volume of a vial can also be 0.01ml, 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8ml, 0.9 ml, 1 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7ml, 1.8 ml, 1.9 ml, 2 ml, 2.1 ml, 2.2 ml, 2.3 ml, 2.4 ml, 2.5 ml, 2.6ml, 2.7 ml, 2.8 rat, 2.9 ml, 3 ml, 3.1 ml, 3.2 ml, 3.3 ml, 3.4 ml, 3.5ml, 3.6 ml, 3.7 ml, 3.8 ml, 3.9 ml, 4 ml, 4.1 ml, 4.2 ml, 4.3 ml, 4.4ml, 4.5 ml, 4.6 ml, 4.7 ml, 4.8 ml, 4.9 ml, 5 ml, 5.1 ml, 5.2 ml, 5.3ml, 5.4 ml, 5.5 ml, 5.6 ml, 5.7 ml, 5.8 ml, 5.9 ml, 6 ml, 6.1 ml, 6.2ml, 6.3 ml, 6.4 ml, 6.5 ml, 6.6 ml, 6.7 ml, 6.8 ml, 6.9 ml, 7 ml, 7.1ml, 7.2 ml, 7.3 ml, 7.4 ml, 7.5 ml, 7.6 ml, 7.7 ml, 7.8 ml, 7.9 ml, 8ml, 8.1 ml, 8.2 ml, 8.3 ml, 8.4 ml, 8.5 ml, 8.6 ml, 8.7 ml, 8.8 ml, 8.9ml, 9 ml, 9.1 ml, 9.2 ml, 9.3 ml, 9.4 ml, 9.5 ml, 9.6 ml, 9.7 ml, 9.8ml, 9.9 ml, or 10 ml. The volume of a vial can also be 0.01 to 0.1 ml,0.11 to 1 ml, 1.1 to 1.11, 1.11 to 1.2, 1.21 to 1.3, 1.31 to 1.4, 1.41to 1.5, 1.51 to 1.6, 1.61 to 1.7, 1.71 to 1.8, 1.81 to 1.9, 1.91 to 2,2.01 to 2.1, 2.11 to 2.2, 2.21 to 2.3, 2.31 to 2.4, 2.41 to 2.5, 2.51 to2.6, 2.61 to 2.7, 2.71 to 2.8, 2.81 to 2.9, 2.91 to 3, 3.01 to 3.1, 3.11to 3.2, 3.21 to 3.3, 3.31 to 3.4, 3.41 to 3.5, 3.51 to 3.6, 3.61 to 3.7,3.71 to 3.8, 3.81 to 3.9, 3.91 to 4, 4.01 to 4.1, 4.11 to 4.2, 4.21 to4.3, 4.31 to 4.4, 4.41 to 4.5, 4.51 to 4.6, 4.61 to 4.7, 4.71 to 4.8,4.81 to 4.9, 4.91 to 5, 5.01 to 5.1, 5.11 to 5.2, 5.21 to 5.3, 5.31 to5.4, 5.41 to 5.5, 5.51 to 5.6, 5.61 to 5.7, 5.71 to 5.8, 5.81 to 5.9,5.91 to 6, 6.01 to 6.1, 6.11 to 6.2 ml, 6.21 to 6.3 ml, 6.31 to 6.4 ml,6.41 to 6.5 ml, 6.51 to 6.6 ml, 6.61 to 6.7 ml, 6.71 to 6.8 ml, 6.81 to6.9 ml, 6.91 to 7 ml, 7.01 to 7.1 ml, 7.11 to 7.2 ml, 7.21 to 7.3 ml,7.31 to 7.4 ml, 7.41 to 7.5 ml, 7.51 to 7.6 ml, 7.61 to 7.7 ml, 7.71 to7.8 ml, 7.81 to 7.9 ml, 7.91 to 8 ml, 8.01 to 8.1 ml, 8.11 to 8.2 ml,8.21 to 8.3 ml, 8.31 to 8.4 ml, 8.41 to 8.5 ml, 8.51 to 8.6 ml, 8.61 to8.7 ml, 8.71 to 8.8 ml, 8.81 to 8.9 ml, 8.91 to 9 ml, 9.01 to 9.1 ml,9.11 to 9.2 ml, 9.21 to 9.3 ml, 9.31 to 9.4 ml, 9.41 to 9.5 ml, 9.51 to9.6 ml, 9.61 to 9.7 ml, 9.71 to 9.8 ml, 9.81 to 9.9 ml, or 9.91 to 10ml.

In some embodiments, pharmaceutical compositions described herein arelyophilized or frozen. Bacterial cells in the lyophilized or frozenpharmaceutical compositions can be stored at −70° C. In someembodiments, the bacterial cells can be stored at 10° C., 4° C., 0° C.,−5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −35° C., −40° C.,−45° C., −50° C., −55° C., −60° C., −65° C., −70° C., −75° C., or −80°C. In other cases, the bacterial cells can also be stored from −80° C.to −70° C., from −70° C. to −60° C., from −60° C. to −50° C., from −50°C. to −40° C., from −40° C. to −30° C., from −30° C. to −20° C., from−20° C. to −10° C., from −10° C. to 0° C., or from 0° C. to 10° C. Insome embodiments, at least 70% of the stored lyophilized or frozenbacterial cells can remain viable after 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 13 months, 14 months, 15 months, 16 months, 17months, 18 months, 19 months, 20 months, 21 months, 22 months, 23months, 24 months, 25 months, 26 months, 27 months, 28 months, 29months, 30 months, 31 months, 32 months, 33 months, 34 months, 35months, or 36 months. In some cases, at least 75% of the storedlyophilized or frozen bacterial cells can remain viable after 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, 25 months, 26 months, 27months, 28 months, 29 months, 30 months, 31 months, 32 months, 33months, 34 months, 35 months, or 36 months. In other cases, at least 80%of the stored lyophilized or frozen bacterial cells can remain viableafter 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months,14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20months, 21 months, 22 months, 23 months, 24 months, 25 months, 26months, 27 months, 28 months, 29 months, 30 months, 31 months, 32months, 33 months, 34 months, 35 months, or 36 months. In someembodiments, at least 85% of the stored lyophilized or frozen bacterialcells can remain viable after 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 13 months, 14 months, 15 months, 16 months, 17 months, 18months, 19 months, 20 months, 21 months, 22 months, 23 months, 24months, 25 months, 26 months, 27 months, 28 months, 29 months, 30months, 31 months, 32 months, 33 months, 34 months, 35 months, or 36months. In other embodiments, at least 90% of the stored lyophilized orfrozen bacterial cells can remain viable after 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10months, 11 months, 12 months, 13 months, 14 months, 15 months, 16months, 17 months, 18 months, 19 months, 20 months, 21 months, 22months, 23 months, 24 months, 25 months, 26 months, 27 months, 28months, 29 months, 30 months, 31 months, 32 months, 33 months, 34months, 35 months, or 36 months. At least 95% of the stored lyophilizedor frozen bacterial cells can also remain viable after 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 13 months, 14 months, 15months, 16 months, 17 months, 18 months, 19 months, 20 months, 21months, 22 months, 23 months, 24 months, 25 months, 26 months, 27months, 28 months, 29 months, 30 months, 31 months, 32 months, 33months, 34 months, 35 months, or 36 months. In some embodiments, atleast 99% of the stored lyophilized or frozen bacterial cells can remainviable after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months,7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13months, 14 months, 15 months, 16 months, 17 months, 18 months, 19months, 20 months, 21 months, 22 months, 23 months, 24 months, 25months, 26 months, 27 months, 28 months, 29 months, 30 months, 31months, 32 months, 33 months, 34 months, 35 months, or 36 months.

A kit of the present disclosure can provide various components for usinga pharmaceutical composition as described herein. Such components caninclude container(s), test sample(s), and/or equipment for analyzing apharmaceutical composition, e.g., its viability, pH of the storagemedium, etc. Thus, kits of this disclosure can allow for user-friendly,accurate and reliable use of a pharmaceutical composition, including,but not limited to dosing, administration, storage, transport. In someembodiments, the pharmaceutical composition comprises a microbialconsortium comprising any one or of the bacterial strain shown in TABLE2. In such instances, the kit can comprise a pharmaceutical compositioncomprising at least one, at least two, or all the strains of TABLE 1.

Methods of Treatment

The present disclosure provides methods for using a pharmaceuticalcomposition described herein for the prevention and/or treatment of adisease. Such diseases can include inflammatory diseases, metabolicdiseases, or autoimmune diseases. Such diseases can be a result of adysbiosis or dysbiosis associated conditions in the subject, or allergicType I hypersensitivity. Such diseases can also include allergic Type IIhypersensitivity, allergic Type III hypersensitivity, or allergic TypeIV hypersensitivity. Such dysbiosis can be dysbiosis of a gut microbiotaof the subject. In some instances, the inflammatory disease is anallergy. In other cases, the inflammatory disease is dermatitis. Suchallergy can be allergic asthma, including allergic pediatric asthma, andfood allergy. Such metabolic diseases can include obesity, diabetes, ora metabolic syndrome.

Thus, in some instances, a pharmaceutical composition described hereincan be formulated for administration to a subject, wherein such subjectcan have or is suspected of having an allergy. Such subject can bemulti-sensitized, e.g., to two or more allergens. Such subject can be amammal. In some instances, the subject is a human. Such pharmaceuticalcomposition, when administered to a subject such as a rodent or a human,can have anti-inflammatory effect(s) useful in the prevention and/ortreatment of inflammatory disease. In some instances, suchanti-inflammatory effect(s) can be elicited when the pharmaceuticalcomposition is orally administered.

In some instances, a subject that is treated using a pharmaceuticalcomposition herein that is a human. The human subject can be a neonate,an infant, a toddler, a child, a teenager, or an adult. In someinstances, the neonate can be less than about 3 days old, less thanabout 1 week old, less than about 2 weeks old, less than about 3 weeksold, less than about 4 weeks old, less than about 8 weeks old. In someinstances, the infant can be at least about 2 months old, at least 6about months old, be at least about 12 about months old. In someinstances, a pharmaceutical composition can be used to treat a subjectthat can be between about 2 and about 18 years old, be at least about 18years old. The subject can be between 2 and 18 years old, or is at least18 years old. The subject can be between about 2 and about 18 years old,or is at least about 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90)years old. In some instances, the subject can be between about 2 andabout 18 years old, or about 19 years old. The subject can be betweenabout 2 and about 18 years old, or about 19 years old. The subject canbe between about 2 and about 18 years old, or about 20 years old. Thesubject can be between about 2 and about 18 years old, or about 20 yearsold. The subject can be between about 2 and about 18 years old, or about25 years old. The subject can be between about 2 and about 18 years old,or about 25 years old. The subject can be between about 2 and about 18years old, or about 30 years old, or older. The subject can be betweenabout 2 and about 18 years old, or about 30 years old. In someembodiments, the subject is from about 18 years to about 40 years ofage, from about 12 years to about 17 years of age, and/or from about 2years to about 11 years of age. A pharmaceutical composition herein canbe mixed with milk breast milk, formula (for nursing an infant), or foodfor administration.

A pharmaceutical composition herein can be administered for variousperiods of time according to different administration schedules. Atreatment period may vary between subjects and individuals and candepend on various factors as described herein, e.g., disease state, age,etc. In some instances, a subject can be treated for one day to at leastabout one week, for about a week to about one month, or for about onemonth to about one year. In such instances, the subject can be treatedfor about one month, two months, or three months. In some cases,treatment can be performed on consecutive days, consecutive weeks,and/or consecutive months. In some embodiments, a pharmaceuticalcomposition is administered for about 28, 29, or 30 consecutive days.

Methods of treatment herein can include administering a pharmaceuticalcomposition of this disclosure once, two, three, four, five, six, seven,eight, nine, ten, eleven, or twelve times daily. In various instances, apharmaceutical composition of this disclosure is administered twicedaily. Such twice daily administration can be performed in the morningand in the evening. In such cases, there can be a period of about 8, 12,or 16 hours between the first and the second administration of a givenday.

In various embodiments herein, the pharmaceutical composition that isadministered to a human subject for prevention and/or treatment of aninflammatory disease such as an allergy comprises at least one of thebacterial strains A. muciniphila (DSM 33213), F. prausnitzii (DSM33185), and/or L. crispatus (DSM 33187) listed in TABLE 1. Suchpharmaceutical composition can be administered to a group subjects(e.g., about 10, 20 or 40 subject) of 2-11, 12-17, and 18-40 years ofage twice daily for about 28 consecutive days. In such instances, suchpharmaceutical composition can be administered in a 1 mL unit dose as aliquid suspension. Such unit dose can be added to cold or roomtemperature food and drinks for administration.

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1: Growth, Isolation, and Characterization of Bacterial Strains

Provided herein are methods for the growth, isolation, andcharacterization of bacterial strains isolated from a human sample. Suchstrains may be used as part of a bacterial consortium described herein.

1. Growth of Bacteria

Generally, the procedures for growing bacteria as described herein canbe used for culturing obligatory and facultative anaerobic bacterialstrains. The bacterial strains in this example were derived from a humanfecal sample and were grown on selective media. Upon subculturing, thecolonies were transferred to a liquid medium and subsequently preparedfor PCR and sequencing. Colonies were also preserved as glycerol stocks.

First, human fecal samples were collected in anaerobic transport media(Anaerobic Systems As-915) or in fecal collection vials sealed within aplastic bag containing an anaerobic atmosphere generating system (e.g.,AnaeroPouch Thermo Fisher R686001). All samples were immediatelytransferred into an anaerobic chamber to minimize transit time andpotential oxygen exposure to ensure viability of the anaerobic strains.

Serial dilution tubes were prepared by aliquoting 0.9 mL of PBS+Cys(1×PBS+0.1% w/w L-cysteine) into 13 tubes (with a volume of 1.5 mL).Using a disposable spatula or loop, 20-30 mg of sample was transferredinto a first 1.5 mL tube containing 0.9 mL of PBS+Cys. The resultingmixture was vortexed for approximately 30 seconds and 0.1 mL of theresulting, homogenous solution was transferred into the second 1.5 mLtube containing 0.9 mL of PBS+Cys. This step was then repeated until all13 tubes contained serial dilutions (e.g., 1-10{circumflex over ( )}−12dilutions in vials 1-13) of the sample.

Using a disposable hockey stick spreader, ˜0.1 mL from the sample tubescontaining the dilutions 10{circumflex over ( )}−5 to 10{circumflex over( )}−12 (vials 6-13) were added to separate agar plates containing theselective agar growth media. The agar plates were then sealed withparafilm to prevent evaporation and placed into an incubator for 72hours at 37° C. Colonies that fit a colony's (e.g., L. crispatus (DSM33187), A. muciniphila (DSM 33213), F. prausnitzii (DSM 33185), etc.)specific morphology were identified, and placed on a new, pre-reducedagar plate for isolation. The agar plates were sealed with parafilm andplaced into an anaerobic incubator for another 72 hours at 37° C.

The isolated colonies were transferred into liquid media by pickingspecifically isolated colonies from culture plates and resuspending thecolonies in 1 mL of pre-reduced liquid broth. Positive and negativecontrols of selected organisms are inoculated in parallel to compare forgrowth and monitor for contamination, respectively. All liquid colonysamples are then incubated for 72 hours at 37° C.

Using the positive and negative controls, positive match broth cultureswere identified. Glycerol stocks of positive match broth cultures wereprepared by transferring 0.75 mL of the broth culture solution into a 2mL cryotube containing 0.75 mL of 50% v/v glycerol in PBS. Sealedcryotube samples were then removed from the anaerobic chamber and storedat −80° C. The remaining broth culture samples were used for isolateidentification using 16S-based PCR as described below.

2. 16S-Based PCR for Isolate Identification

Broth culture samples were centrifuged to form cell pellets and theresulting supernatant is carefully removed to leave the formed cellpellet intact. The cell pellet was then resuspended in 0.5-1 mLultrapure water.

The PCR Mastermix for a final reaction volume of 50 μL was preparedusing the following PCR components (NEB E5000S) and volumes: 10× Buffer(5 μL), 10 mM dNTPs (1 μL), 10 μM 27F Forward Primer (1 μL), 10 μM 1492RReverse Primer (1 μL), Tag Polymerase (0.25 μL), and sterile water(40.25 μL). The PCR Mastermix (48.5 μL) and 1.5 μL of resuspendedbacterial cells were placed into a 0.2 mL PCR strip tube and vortexedbefore the PCR reaction samples were exposed to the followingthermocycler protocol (TABLE 3):

TABLE 3 PCR Thermocycler Protocol. Step Temp Time 1 95° C. 2 min 2 95°C. 30 sec 3 50° C. 30 sec 4 68° C. 1 min 30 sec (30 repeats of steps2-4) 5 72° C. 5 min 6  4° C. HOLD

Upon completion of the PCR reactions, samples were submitted for Sangersequencing using GENEWIZ or a comparable vendor.

3. Characterization of Isolated Bacterial Strains

TABLE 4 below shows short chain fatty acid production, antibioticresistance, and whole genome sequencing analysis of the strains (alsoshown in TABLE 2) Akkermansia muciniphila (DSM 33213), Faecalibacteriumprausnitzii (DSM 33185), and Lactobacillus crispatus (DSM 33187) that,in various embodiments of this disclosure, can form a bacterialconsortium for use in a pharmaceutical composition.

TABLE 4 Isolate Characterization. Short Chain Fatty Acid AntibioticWhole Production Resistance Genome Genus Species Strain DesignationAnalysis Analysis Seq. Akkermansia muciniphila A. muciniphila Yes YesYes (DSM 33213) Faecalibacterium prausnitzii F. prausnitzii Yes Yes Yes(DSM 33185) Lactobacillus crispatus L. crispatus Yes Yes Yes (DSM 33187)

Example 2: Manufacturing of a Bacterial Composition A

Manufacturing conditions were generated to increase yield and growthrate of bacterial strains described herein. In particular, manufacturingconditions for increased yield and growth rate were obtained for thebacterial strains Akkermansia muciniphila (DSM 33213), Faecalibacteriumprausnitzii (DSM 33185), and Lactobacillus crispatus (DSM 33187). FIG. 1herein provides a schematic flow chart summarizing the manufacturingsteps for preparing Composition A using these strains.

1. Media Preparation

Animal-free culture media was used in the manufacturing steps of thisexample. To produce bacterial batches, 5 L of broth media was firstprepared for each of the strains. After completely dissolving mediacomponents by vigorous stirring for 15 mins in USP grade water, the pHof the media was adjusted with hydrochloric acid. The pH adjusted mediawas then transferred to a biosafety cabinet and filter sterilized using0.2 μm vacuum filter units (e.g., using 5 times 1-liter portions).Filter sterilized growth media was immediately transferred to ananaerobic chamber containing an atmosphere of N₂H₂CO₂ (90:5:5) andstored with a vented cap for 12-18 hours to reduce prior to inoculation.

2. Inoculum and Culture

After reduction of the media under anaerobic conditions for 12-18 hours,10 mL from each 1 L filter flask was transferred to a sterilepre-reduced 15 mL screw cap tube and labeled “Sterility Control # of 5”.An additional 40 mL of sterile media was transferred to two separate 50mL Falcon tubes labeled “Starter Culture X of 2”. One 2 mL master cellbank (MCB) cryovial was removed from −70° C. storage and the exterior ofthe vial was cleaned with 70% EtOH and wiped dry with a lint-free wipe.The MCB aliquot was then transferred into the anaerobic chamber andplaced in a tube rack to thaw for 5-10 mins. Once completely thawed, asterile 1 mL filtered pipette tip was used to transfer 500 μL of thethawed 1 mL MCB aliquot into each of the two 40 mL starter cultures. Thecaps on the inoculated starter culture tubes and sterility control weresecurely tightened and the controls and starter cultures are grown atabout 37° C. for 12-16 hours under anaerobic conditions.

After 12-16 hours, the starter cultures and sterility control wereremoved from the incubator and visibly inspected for growth (e.g.,turbidity). Additionally, lack of visible growth or turbidity wasconfirmed in the sterility controls prior to proceeding. Once confirmed,a sterile 10 mL serological pipette tip was used to carefully transfer10 mL of the starter culture to each of the five pre-warmed 1 L flaskscontaining pre-reduced, filter sterilized culture media. The cultureswere then incubated for 12-16 hours at 37° C. under anaerobicconditions. After incubation, the turbidity of the cultures wasquantified using absorbance spectrometry (Epoch 2 Plate Reader, Biotek)to confirm growth within set parameters. Once the culture absorbance wasconfirmed to fall within the target OD₆₀₀ range, the cells wereharvested by centrifugation.

3. Harvest

Twelve to 18 hours prior to cell harvest, five sterile 1 L centrifugebottles (Beckman Coulter) with screw cap seals were placed in theanaerobic chamber to allow the bottles to reduce prior to use. Afterculture growth and validation that cultures fall within target OD₆₀₀range, the cultures were transferred into sterile 1 L centrifugebottles. The caps on the centrifuge bottles were tightly sealed toprevent gas exchange and the bottles were transferred to a pre-cooledfloor centrifuge containing a 6×1 L rotor. Cells were pelleted at8,000×g for 30 mins at 4° C. After centrifugation, the cell pellets weretransferred back into the anaerobic chamber and the clarified culturesupernatant was completely removed from the pellets using a sterileserological pipette. The cell pellets were then combined by resuspensionin a 40× concentration in a pre-reduced cryoprotectant solution. Theconcentrated cell suspensions were then dispensed into 1 mL aliquots inpre-reduced, pre-labeled 2 mL screw cap cryovials and immediatelytransferred to pre-labeled storage boxes at −70° C. Drug substancestrains underwent specification testing three days after manufacturingand initial storage at −70° C.

Described below are production procedures for A. muciniphila (DSM33213), F. prausnitzii (DSM 33185), and L. crispatus (DSM 33187) cellsused in a pharmaceutical composition.

A. Generation of A. muciniphila (DSM 33213) Cells for Use in aPharmaceutical Composition

For preparation of the culture medium, in a 5 L beaker, 4.9 L water (forinjection) was added before the addition, under mixing, of Difco™ SelectSoytone (82.5±0.82 g), Bacto™ Yeast Extract (12.5±0.12 g), SodiumBicarbonate (NaHCO₃) (5±0.05 g), Dibasic Potassium Phosphate (K₂HPO₄)(12.5±0.12 g), Sodium Chloride (NaCl) (1.5±0.015 g), Magnesium SulfateHeptahydrate (MgSO₄×7 H₂O) (0.5±0.05 g), Calcium Chloride (CaCl₂)(0.5±0.05 g), Glucose (Dextrose) (22.6±0.22 g), N-acetylglucosamine(27.7±0.27 g), L-Threonine (20±0.2 g), and L-Cysteine (5±0.05 g). Themixture was stirred until all components are fully dissolved, clear andfree of solids and precipitates.

The pH of the NAGT media was adjusted to 6.5±0.1. The medium was thenvacuum filtered and divided into 5 1 L batches. After the culture mediaare completely reduced (after approximately 12-16 hours), startercultures of A. muciniphila (DSM 33213) bacterial cells were preparedwith a 45 mL volume of medium. A. muciniphila (DSM 33213) bacterialcells (approximately 500 μL of stock solution) were removed from thecell bank and added into the prepared vials containing growth medium.The cell suspensions are then allowed to warm and incubated for 24-60hours in a 37° C. incubator unit, resulting in a turbid/cloudysuspension. Subsequently, the absorbance at 600 nm is measured andrecorded in triplicates to achieve a value of about 0.7-1.1.

The culture flasks were then centrifuged using a JLA8.1000 centrifuge at8000 rpm, for 30 min at 4° C. After the supernatants were removed, theresidual cell pellets were re-suspended in 25 mL of sterile PBS-GC (40×concentration of the original culture volume), and then combined toyield a homogenous solution. The A. muciniphila (DSM 33213) cellsuspension was aliquoted into 2 mL cryovials to achieve a final A.muciniphila (DSM 33213) cell concentration of >1×10{circumflex over( )}9 live A. muciniphila (DSM 33213) cells per vial and was stored at−80° C. until further use.

B. Generation of F. prausnitzii (DSM 33185) Cells for Use in aPharmaceutical Composition

For preparation of the YFAP Vitamin Mix Solution, a 1 L bottle is filledwith water (for injection) followed by the addition, under mixing, ofbiotin (10±1 mg), cobalamin (10±1 mg), p-aminobenzoic acid (30±1 mg),folic acid (50±1 mg), pyridoxamine (150±1 mg), thiamine (50±1 mg), andriboflavin (50±1 mg). The mixture was stirred until all components werefully dissolved, clear and free of solids and precipitates.Subsequently, the YFAP Vitamin mix medium was filtered and sterilized.

For preparation of the culture medium, in a 5 L beaker, 4.9 L water (forinjection) was added before the addition, under mixing, of BBL™ PhytonePeptone (50±0.5 g), Difco™ Select Soytone (50±0.5 g), Bacto™ YeastExtract (25±0.25 g), sodium bicarbonate (NaHCO₃) (5±0.05 g), dibasicpotassium phosphate (K₂HPO₄) (12.5±0.12 g), sodium chloride (NaCl)(5±0.05 g), magnesium sulfate heptahydrate (MgSO₄×7 H₂O) (1±0.01 g),sodium acetate (NaOAc) (25±0.25 g), glucose (dextrose) (50±0.25 g),sodium propionate (5±0.05 g), L-cysteine (5±0.05 g), and YFAP VitaminMix Solution (1 mL, prepared as described above). The mixture wasstirred until all components were fully dissolved, clear and free ofsolids and precipitates.

The pH of the YFAP medium was then adjusted to 6.5 using 5 Mhydrochloride solution. The medium was then filtered and allow to standin the dark for about 12-18 hours for full reduction to an anaerobicstate. After a certain volume of medium was transferred to the starterculture tube, approximately 500 μL of the stock F. prausnitzii (DSM33185) solution was transferred into the starter culture tube andincubated at 37° C. for about 12-16 hours. The starter culture tube wasevaluated for turbidity and divided into 5 aliquots and added topre-warmed 1 L flask containing sterile culture media, followed byincubation for 12-16 hours at 37° C.

After incubation, the absorbance at 600 nm was determined and repeatedin triplicates to ensure absorbance falls within the range of 1.4-1.8absorbance. The culture flasks were centrifuged using a JLA8.1000centrifuge at 8000 rpm, for 30 min at 4° C. After the supernatants wereremoved, the residual cell pellets were re-suspended in 25 mL of sterilePBS-GC (40× concentration of the original culture volume), and thencombined to yield a homogenous solution. The F. prausnitzii (DSM 33185)cell suspension was aliquoted into 2 mL cryovials to achieve a final F.prausnitzii (DSM 33185) cell concentration of >1×10{circumflex over( )}9 live F. prausnitzii (DSM 33185) cells per vial and was stored at−80° C. until further use.

C. Generation of L. crispatus (DSM 33187) Cells for Use in aPharmaceutical Composition

For preparation of the vMRS media, in a 5 L beaker, 4.9 L water (forinjection) was added before the addition, under mixing, of 273 g of vMRSpowder and dipotassium phosphate (K₂HPO₄) (12.5±0.1 g). The mixture wasstirred until all components were fully dissolved, clear and free ofsolids and precipitates.

The pH of the vMRS media was then adjusted to 6.5 with NH₄OH or aceticacid. The medium was then filtered using 0.2 μm vacuum filter units andallow to stand in the dark for about 12-18 hours for full reduction toan anaerobic state. After a certain volume (e.g., 45 mL) of medium wastransferred to the starter culture tube, approximately 500 μl of thestock L. crispatus (DSM 33187) solution was transferred into the starterculture tube and incubated at 37° C. for about 16-20 hours. The starterculture tube was evaluated for turbidity, divided into 5 aliquots andadded to the pre-warmed 1 L flask containing sterile culture media,followed by incubation for 16-20 hours at 37° C.

After incubation, the absorbance at 600 nm was determined and repeatedin triplicates to ensure absorbance falls within the range of 1.0-1.4absorbance. The culture flasks were centrifuged using a JLA8.1000centrifuge at 8000 rpm, for 20 min at 4° C. After the supernatants wereremoved, the residual cell pellets were re-suspended in 25 mL of sterilePBS-GC (40× concentration of the original culture volume), and thencombined to yield a homogenous solution. The L. crispatus (DSM 33187)cell suspension is aliquoted into 1 mL cryovials to achieve a final L.crispatus (DSM 33187) cell concentration of >1×10{circumflex over ( )}9live L. crispatus (DSM 33187) cells per vial and was stored at −80° C.until further use.

D. Assembly of Composition A

In order to prepare Composition A that may be used as a bacterialconsortium in a pharmaceutical composition described herein, and thatincludes the three bacterial strains A. muciniphila (DSM 33213), F.prausnitzii (DSM 33185), and L. crispatus (DSM 33187), the number ofmetabolically active bacterial cells in the finalized cell suspensionsstored at −80° C. in cryovials was determined for each strain. Themaximum number of potential oral doses that can be produced from thethree strain batches can be calculated as follows (TABLE 5):

TABLE 5 Calculation of Potential Doses from Prepared Strain Batches.Bacterial strain Formula A. muciniphila Number of Potential Doses fromA. muciniphila (DSM 33213) batch = (DSM 33213) ((A. muciniphila (DSM33213) Average Potency in CFU/mL) X (F. prausnitzii (DSM 33185) BatchVolume in mL))/(1 × 10^(∧)9 CFU/dose) F. prausnitzii Number of PotentialDoses from F. prausnitzii (DSM 33185) batch = (DSM 33185) ((F.prausnitzii (DSM 33185) Average Potency in CFU/mL) X (F. prausnitzii(DSM 33185) Batch Volume in mL))/(4 × 10^(∧)8 CFU/dose) L. crispatusNumber of Potential Doses from L. crispatus (DSM 33187) batch = (DSM33187) ((L. crispatus (DSM 33187) Average Potency in CFU/mL) X (L.crispatus (DSM 33187) Batch Volume in mL))/(4 × 10^(∧)8 CFU/dose)

A full ingredient list for Composition A is provided in TABLE 6:

TABLE 6 Components of Composition A. Absolute or Component Type relativeamount Bacterial strain A. muciniphila 5 × 10^(∧)8 CFU (DSM 33213)Bacterial strain F. prausnitzii 5 × 10^(∧)8 CFU (DSM 33185) Bacterialstrain L. crispatus 5 × 10^(∧)8 CFU (DSM 33187) Cryoprotectant Glycerol20% (v/v) Antioxidant L-cysteine 0.1% (w/w) Buffer PBS 0.5-1 mL

Once the stock vials containing the strain listed in TABLE 6 had beenthawed (about 15-20 minutes after removal from freezer), the calculatedamounts of cell suspension volumes for each strain were transferred to a1 L glass bottle and the volume increased using PBS-GC to arrive at thecalculated concentration of cells per mL (e.g., 5×10{circumflex over( )}8 CFU/bacterial species/mL in this example). The resultinghomogenous suspension of mixed species drug product was aliquoted into 1mL volumes into cryovials and stored at −80° C. until further use.

E. Quality Control of Composition A

The following parameters, test methods and specifications were evaluatedfor each batch of Composition A for quality control purposes asdescribed in TABLE 7.

TABLE 7 Exemplary Quality Control Parameters for Composition A.Parameter tested Assay method used Result Concentration of ViableDilution plating on yBHI Agar 1 × 10^(∧)8-5 × 10^(∧)10 F. prausnitzii(DSM 33185) for F. prausnitzii CFU/mL Colony Morphology of Visualinspection of colonies Circular, entire margin, F. prausnitzii (DSM33185) growing on yBHI Agar flat, small-moderate size, cream-tan color.Concentration of Viable Dilution plating on Mucin 1 × 10^(∧)8-5 ×10^(∧)10 A. muciniphila (DSM 33213) Agar for A. muciniphila CFU/mLColony Morphology of Visual Inspection of colonies Circular, entiremargin, A. muciniphila (DSM 33213) growing on Mucin Agar raised,punctiform size, opaque-translucent. Concentration of Viable Dilutionplating on MRS Agar 1 × 10^(∧)8-5 × 10^(∧)10 L. crispatus (DSM 33187)for L. crispatus growth CFU/mL Colony Morphology of Visual Inspection ofcolonies Circular, entire margin, L. crispatus (DSM 33187) growing onMRS Agar raised, small-moderate, white-cream color. SpecifiedMicroorganisms Test for objectionable Absent (E.coli, C. albicans,microorganisms according Salmonella, S. aureus, P. to USP <62> Bioburden(TAMC/TYMC) Enumeration of TAMC < 200, TYMC < 20 microorganismsaccording to USP <61>

The test for objectionable microorganisms was performed according toUSP<62> using standard protocol. Briefly, the sample was first enrichedby inoculating in Soybean Casein Digest Broth (SCDA), or otherappropriate neutralizing media, and then streaked onto selective agarsfor determination of the presence of specified/objectionablemicroorganisms.

The total number of microorganisms present in a sample was performedaccording to USP<61> using standard protocol. Such enumeration ofmicroorganisms was carried out using either membrane filtration, pourplating, or the spread plate method.

Example 3: Growth Media Optimization

Provided in this example is an optimization of growth media compositionfor culturing bacterial strains described herein. In particular, suchstrains include Akkermansia muciniphila (DSM 33213), Faecalibacteriumprausnitzii (DSM 33185), and Lactobacillus crispatus (DSM 33187) for usein a bacterial consortium.

1. Akkermansia muciniphila (DSM 33213)

Growth of A. muciniphila (DSM 33213) cells in N-acetylglucosaminethreonine (NAGT) media was evaluated at different pH values, along withremoving one media component at a time to evaluate the effect of suchmedia components on the growth of A. muciniphila (DSM 33213) cells.General NAGT media had a pH of 6.5 and contained 11.9 mM sodiumbicarbonate. A ˜40% increase in growth (as measured by OD₆₀₀, curve #2,FIG. 2) was observed when A. muciniphila (DSM 33213) cells were grown inmodified NAGT media with a pH of 7.5 containing 47.6 mM sodiumbicarbonate. The increase in biomass was also confirmed by using theplating method. Eliminating glucose (curve #3, FIG. 2), magnesiumsulfate (curve #4, FIG. 2) or calcium chloride (curve #5, FIG. 2) fromthe modified NAGT media showed little to no effect in the growth of A.muciniphila (DSM 33213) cells.

FIG. 3 shows that soytone and N-acetyl glucosamine (NAG) appear to benecessary for growth of A. muciniphila (DSM 33213) cells, as shown bygrowth curves #2 and #3, respectively, that demonstrate no bacterialgrowth in media lacking soytone or NAG. Growth curve #1 shows stronggrowth of A. muciniphila (DSM 33213) cells in NAGT media containing bothsoytone and NAG.

2. Faecalibacterium prausnitzii (DSM 33185)

The effect of certain components in YFAP medium on the growth of F.prausnitzii (DSM 33185) cells was evaluated by removing one mediacomponent at a time. For example, FIG. 4 shows that yeast extract (e.g.,curve #3 shows FP (DSM 33185) growth in media lacking yeast extract) andcysteine (e.g., curve #4 shows FP (DSM 33185) growth in media lackingyeast extract) appear to be necessary for growth of F. prausnitzii (DSM33185) cells when compared to growth of F. prausnitzii (DSM 33185) cellsin the complete medium (curve #1). Absence of sodium acetate (curve #5)appears to reduce bit not prevent the growth of F. prausnitzii (DSM33185) cells, whereas absence of soytone (curve #2) appears to lead tocell death during late log and stationary phases.

The impact for types of vitamin supplement for the growth F. prausnitzii(DSM 33185) cells was also examined. For example, FIG. 5 shows that theaddition of vitamins increased the yield of F. prausnitzii (DSM 33185)cells sub-cultured three times in YFAP media without vitamins. The finalOD₆₀₀ of F. prausnitzii (DSM 33185) growing in YFAP with vitamins(YFAP+vitamin) was about 10% higher than those growing in YFAP medialacking thiamine (YFAP No thiamine), pyroxamine (YFAP No pyroxamine),folic acid (YFAP No folic acid), cobalamin (YFAP No cobalamin), PABA(YFAP No PABA), riboflavin (YFAP No riboflavin), vitamins (YFAP Novitamins), or biotin (YFAP No biotin). The YFAP+vitamin media comprisesthe YFAP media added with a complete vitamin mix solution (e.g., YFAPvitamin mix). The YFAP vitamin comprises about 10 mg/L of biotin, about10 mg/L of cobalamin, about 30 mg/L of p-aminobenzoic acid, about 50mg/L of folic acid, about 150 mg/L of pyridoxamine, about 50 mg/L ofthiamine, and about 50 mg/L of riboflavin.

In another example, FIG. 6 shows that cobalamin was a notable factor forthe optimal growth of F. prausnitzii (DSM 33185) cells sub-cultured onetime in YFAP media without vitamins. The final OD₆₀₀ of F. prausnitzii(DSM 33185) growing in YFAP lacking cobalamin (YFAP No cobalamin) wasabout 30% lower than those growing in YFAP media lacking thiamine (YFAPNo thiamine), pyroxamine (YFAP No pyroxamine), folic acid (YFAP No folicacid), PABA (YFAP No PABA), riboflavin (YFAP No riboflavin), biotin(YFAP No biotin) or YFAP media with the addition of vitamins(YFAP+vitamins). The growth disadvantage of YFAP lacking cobalamin wassimilar to that of YFAP lacking vitamins (YFAP No vitamins).

The pH of the culture media was also shown to be a notable factor forthe growth of F. prausnitzii (DSM 33185) cells. FIG. 7 shows the growthof F. prausnitzii (DSM 33185) cells without pH control while FIG. 8shows the growth of F. prausnitzii (DSM 33185) cells with pH control.When pH was controlled at 6 with ammonium hydroxide (NH₄OH), the redoxdropped to −460 mV while the bacterial growth plateaued at aroundOD₆₀₀=0.5. When pH was not controlled, the redox maintained at −400 mV,and the bacterial growth reached OD₆₀₀=1.1. Therefore, the culturewithout pH control yielded >50% more bacteria than that with pH control.

3. Lactobacillus crispatus (DSM 33187)

L. crispatus (DSM 33187) was generally grown in vMRS broth obtained fromHiMedia Laboratories. However, FIG. 9 shows that growth of L. crispatus(DSM 33187) cells was increased about 55% when media was made withBoullion MRS Vegetal (curve #2) obtained from Biokar diagnostics (Ref:BK176HA) compared to use of HiMedia vMRS broth (curve #1). Without beingbound by any theory it is assumed that the observed improvement in cellgrowth is due to a higher amount (about twice the amount with 20 g/L) ofvegetable peptone of the Boullion MRS Vegetal compared to HiMedia vMRSbroth.

Example 4: Large-Scale Growth of Bacteria (20 L)

Manufacturing conditions for growing bacteria in 20 L volume of culturewere generated to increase yield and growth rate of bacterial strainsdescribed herein. In particular, manufacturing conditions for increasedyield and growth rate were obtained for the bacterial strainsAkkermansia muciniphila (DSM 33213), Faecalibacterium prausnitzii (DSM33185), and Lactobacillus crispatus (DSM 33187).

Large-Scale Growth of Akkermansia muciniphila (DSM 33213)

To prepare the NAGT medium for 1 L inoculum culture and 20 L primaryculture, medium components are weighted as followings: pea peptone (16.5g/L), yeast extract (2.5 g/L), dextrose (4.52 g/L), dibasic potassiumphosphate (K₂HPO₄) (2.5 g/L), sodium chloride (NaCl) (0.3 g/L),magnesium sulfate heptahydrate (MgSO₄×7 H₂O) (0.1 g/L), sodiumbicarbonate (NaHCO₃) (1 g/L), calcium chloride (0.1 g/L),N-acetylglucosamine (5.54 g/L), L-threonine (4 g/L), L-cysteine (1 g/L).The mixture was stirred until all components are fully dissolved, clearand free of solids and precipitates.

The pH of the NAGT medium was adjusted to 6.5±0.1 with NH₄OH and aceticacid. The medium was then sterilized by autoclaving at 121° C. for 20minutes. Glucose and N-acetylglucosamine feed was filter-sterilizedseparately. The media components were then mixed together.

Inoculum flask containing NAGT was moved to the anaerobic chamber anddegassed with N₂H₂CO₂ (90:5:5) by incubation under anaerobic atmospherefor 48 hours. Inoculum culture was inoculated from the RCB or MCB (0.4%for A. muciniphila (DSM 33213) and incubated under anaerobic atmosphereat 37° C. with no agitation. Culture glucose consumption and opticaldensity were monitored every 2 hours. The culture was stopped if a) aphase of deceleration is observed or b) the culture has been growing for24 hours.

20 L media was degassed by sparging using N₂H₂CO₂ (90:5:5). Degassingwas performed at 6.5 L/min until the redox value dropped and stabilized.Primary culture was inoculated at 5% (eg. ˜1 L of inoculum culture), andculture glucose consumption and optical density were monitored every 2hours. The culture was stirred at 100 rpm and grew at 37° C. As shown inFIG. 10, a feed of 90.4 g of glucose and 110.8 g of N-acetylglucosaminewas added at 24^(th) hour and 48^(th) hour to allow the bacterialculture to maintain in the exponential phase between 25^(th) to 50^(th)hour and enter the stationary phase after 50^(th) hour. The culture wasstopped if a) a phase of growth deceleration was observed or b) theculture had grown for about 70 hours.

Large-Scale Growth of Lactobacillus crispatus (DSM 33187)

To prepare the Vegitone MRS medium for 1 L inoculum culture and 150 Lprimary culture, medium components are weighted as followings: peapeptone (20 g/L), yeast extract (10 g/L), dextrose (20 g/L), dibasicpotassium phosphate (K₂HPO₄) (2.5 g/L), ammonium citrate (0.3 g/L),magnesium sulfate heptahydrate (MgSO₄×7 H₂O) (0.1 g/L), sodium acetate(NaOAc) (5.54 g/L), Tween 80 (4 g/L). The mixture was stirred until allcomponents are fully dissolved, clear and free of solids andprecipitates.

The pH of the Vegitone MRS medium was adjusted to 6.5±0.1 with NH₄OH oracetic acid. The medium was then sterilized by autoclaving at 121° C.for 20 minutes. Glucose sugar solution was filter-sterilized. The mediacomponents were then mixed together.

Inoculum flask containing Vegitone MRS was moved to the anaerobicchamber and degassed with N₂H₂CO₂ by incubation under anaerobicatmosphere for 48 hours. Inoculum culture was inoculated from the RCB orMCB (0.4% for L. crispatus (DSM 33187) and incubated under anaerobicatmosphere at 37° C. with no agitation. Culture glucose consumption andoptical density were monitored every 2 hours. The culture was stopped ifa) a phase of deceleration is observed or b) the culture has beengrowing for 24 hours.

20 L media was degassed by sparging using N₂H₂CO₂ (90:5:5). Degassingwas performed at 6.5 L/min until the redox value dropped and stabilized.Primary culture was inoculated at 1% (eg. ˜200 mL of inoculum culture),and culture glucose consumption and optical density were monitored every2 hours. The culture was stirred at 100 rpm and grew at 37° C. As shownin FIG. 11, a feed of 200 g of glucose was added at the 10^(th) hour and11^(th) hour to allow bacterial culture to maintain in the exponentialphase between 11^(th) to 14^(th) hour and enter the stationary phaseafter 14^(th) hour. The culture was stopped if a) a phase of growthdeceleration was observed or b) the culture had grown for 16 hours.

Large-Scale Growth of Faecalibacterium Prausnitzii (DSM 33185)

To prepare the YFAP medium for 1 L inoculum culture and 20 L primaryculture, media components are weighted as followings: pea peptone (20g/L), yeast extract (5 g/L), dextrose (10 g/L), dibasic potassiumphosphate (K₂HPO₄) (2.5 g/L), sodium chloride (NaCl) (1 g/L), magnesiumsulfate heptahydrate (MgSO₄×7 H₂O) (0.2 g/L), sodium bicarbonate(NaHCO₃) (1 g/L), sodium acetate NaOAc) (5 g/L), L-cysteine (1 g/L).YFAP Vitamin Mix Solution was prepared as described in Example 3. Themixture was stirred until all components are fully dissolved, clear andfree of solids and precipitates.

The pH of the YFAP media was adjusted to 6.5±0.1 with NaOH. The mediumwas then sterilized by autoclaving at 121° C. for 20 minutes. Glucoseand YFAP Vitamin Mix Solution were filter sterilized (0.2 μm filter).The medium components were then mixed together.

Inoculum flask containing YFAP was moved to the anaerobic chamber anddegassed with N₂H₂CO₂ by incubation under anaerobic atmosphere for atleast 16 hours. Inoculum culture was inoculated from the Research CellBank (RCB) or Master Cell Bank (MCB) (0.4% for F. prausnitzii (DSM33185) and incubated under anaerobic atmosphere at 37° C. with noagitation. Culture glucose consumption and optical density weremonitored every 2 hours. The culture was stopped after the culture hasbeen grown for 10-16 hours.

All open vessel operations were performed in either the BiologicalSafety Cabinet (BSC) or Anaerobic Chamber (AC) using good aseptictechniques. 20 L medium was degassed by sparging using N₂H₂CO₂ (90:5:5).Degassing was performed at 6.5 L/min until the redox value dropped andstabilized. Primary culture was inoculated at 1% (e.g., —200 mL ofinoculum culture), and culture glucose consumption and optical densitywere monitored every 2 hours. The culture was stirred at 100 rpm andgrew at 37° C. As shown in FIG. 12, a feed of 200 g of glucose was addedat 9^(th) hour to allow the cell to maintain growth from 9^(th) to14^(th) hour. The culture was stopped if a) a phase of growthdeceleration was observed or b) the culture had grown for 16 hours

Example 5: Large-Scale Growth of Bacteria (150 L)

Manufacturing conditions for growing bacteria in 150 L volume of culturewere generated to increase yield and growth rate of bacterial strainsdescribed herein. In particular, manufacturing conditions for increasedyield and growth rate were obtained for the bacterial strainsAkkermansia muciniphila (DSM 33213), Faecalibacterium prausnitzii (DSM33185), and Lactobacillus crispatus (DSM 33187).

Large-Scale Growth of Akkermansia muciniphila (DSM 33213)

To prepare the NAGT medium for 1 L inoculum culture and 150 L primaryculture, medium components are weighted as followings: pea peptone (16.5g/L), yeast extract (2.5 g/L), dextrose (4.52 g/L), dibasic potassiumphosphate (K₂HPO₄) (2.5 g/L), sodium chloride (NaCl) (0.3 g/L),magnesium sulfate heptahydrate (MgSO₄×7H₂O) (0.1 g/L), sodiumbicarbonate (NaHCO₃) (1 g/L), calcium chloride (0.1 g/L),N-acetylglucosamine (5.54 g/L), L-threonine (4 g/L), L-cysteine (1 g/L).The mixture was stirred until all components are fully dissolved, clearand free of solids and precipitates.

The pH of the NAGT medium was adjusted to 6.5±0.1 with NH₄OH or aceticacid. The medium was then sterilized by autoclaving at 121° C. for 20minutes. Glucose was filter sterilized (0.2 μm). The media componentswere then mixed together.

Inoculum flask containing NAGT was moved to the anaerobic chamber anddegassed with N₂H₂CO₂ by incubation under anaerobic atmosphere for 48hours. Inoculum culture was inoculated from the RCB or MCB (0.4% for A.muciniphila (DSM 33213) and incubated under anaerobic atmosphere at 37°C. with no agitation. Culture glucose consumption and optical densitywere monitored every 2 hours. The culture was stopped if a) a phase ofdeceleration is observed or b) the culture had been growing for 48hours.

150 L media was degassed by sparging using N₂H₂CO₂ (90:5:5). Degassingwas performed at 6.5 L/min until the redox value dropped and stabilized.Primary culture was inoculated at 0.4% (e.g., —600 mL of inoculumculture), and culture glucose consumption and optical density weremonitored every 2 hours. The culture was stirred at 100 rpm and grew at37° C. As shown in FIG. 13, a filter sterilized feed of 678 g of glucoseand 831 g of N-acetylglucosamine was added at 19^(th) hour to allow thebacterial culture to maintain in the exponential growth after 20^(th)hour. The culture was stopped if a) a phase of growth deceleration wasobserved or b) the culture had grown for 24 hours.

Large-Scale Growth of Lactobacillus crispatus (DSM 33187)

To prepare the Vegitone MRS medium for 1 L inoculum culture and 150 Lprimary culture, medium components are weighted as followings: peapeptone (20 g/L), yeast extract (10 g/L), dextrose (20 g/L), dibasicpotassium phosphate (K₂HPO₄) (2.5 g/L), ammonium citrate (0.3 g/L),magnesium sulfate heptahydrate (MgSO₄×7 H₂O) (0.1 g/L), sodium acetate(NaOAc) (5.54 g/L), Tween 80 (4 g/L). The mixture was stirred until allcomponents are fully dissolved, clear and free of solids andprecipitates.

The pH of the Vegitone MRS medium was adjusted to 6.5±0.1 with NH₄OH oracetic acid. The medium was then sterilized by autoclaving at 121° C.for 20 minutes. Glucose was filter sterilized separately. The mediacomponents were then mixed together.

Inoculum flask containing Vegitone MRS was moved to the anaerobicchamber and degassed with N₂H₂CO₂ by incubation under anaerobicatmosphere for 48 hours. Inoculum culture was inoculated from the RCB orMCB (0.4% for L. crispatus (DSM 33187) and incubated under anaerobicatmosphere at 37° C. with no agitation. Culture glucose consumption andoptical density were monitored every 2 hours. The culture was stopped ifa) a phase of deceleration is observed or b) the culture has beengrowing for 24 hours.

150 L media was degassed by sparging using N₂H₂CO₂ (90:5:5). Degassingwas performed at 6.5 L/min until the redox value dropped and stabilized.Primary culture was inoculated at 0.4% (e.g., —600 mL of inoculumculture), and culture glucose consumption and optical density weremonitored every 2 hours. The culture was stirred at 100 rpm and grew at37° C. As shown in FIG. 14, a feed of 5250 g of glucose was added at10^(th) hour and allowed the bacterial culture to maintain in theexponential growth from 10^(th) to 12^(th) hour and enter stationarygrowth after 12^(th) hour. The culture was stopped if a) a phase ofgrowth deceleration was observed or b) the culture had grown for 24hours.

Large-Scale Growth of Faecalibacterium Prausnitzii (DSM 33185)

To prepare the YFAP medium for 1 L inoculum culture and 150 L primaryculture, media components are weighted as followings: pea peptone (20g/L), yeast extract (5 g/L), dextrose (10 g/L), dibasic potassiumphosphate (K₂HPO₄) (2.5 g/L), sodium chloride (NaCl) (1 g/L), magnesiumsulfate heptahydrate (MgSO₄×7 H₂O) (0.2 g/L), sodium bicarbonate(NaHCO₃) (1 g/L), sodium acetate NaOAc) (5 g/L), L-cysteine (1 g/L).YFAP Vitamin Mix Solution was prepared as described in Example 3. Themixture was stirred until all components are fully dissolved, clear andfree of solids and precipitates.

The pH of the YFAP medium was adjusted to 6.5±0.1 with NaOH and aceticacid. The medium was then sterilized by autoclaving at 121° C. for 20minutes. Glucose and YFAP Vitamin Mix Solution were filter sterilized(0.2 μm filter). The media components were then mixed together.

Inoculum flask containing YFAP was moved to the anaerobic chamber anddegassed with N₂H₂CO₂ by incubation under anaerobic atmosphere for atleast 16 hours. Inoculum culture was inoculated from the RCB or MCB(0.4% for F. prausnitzii (DSM 33185) and incubated under anaerobicatmosphere at 37° C. with no agitation. Culture glucose consumption andoptical density were monitored every 2 hours. The culture was stopped ifa) a phase of deceleration is observed or b) the culture had beengrowing for 24 hours.

150 L media was degassed by sparging using N₂H₂CO₂ (90:5:5). Degassingwas performed at 6.5 L/min until the redox value dropped and stabilized.As shown in FIG. 15, primary culture was inoculated at 0.4% (e.g., —600mL of inoculum culture) and maintained exponential growth from 11^(th)to 16^(th) hour, and culture glucose consumption and optical densitywere monitored every 2 hours. The culture was stirred at 100 rpm andgrew at 37° C. As shown in FIG. 16, a feed of 150 g of glucose was addedat 8^(th) hour allowed the bacterial culture to maintain exponentialgrowth after 8^(th) hour. The culture was stopped if a) a phase ofgrowth deceleration was observed or b) the culture had grown for 24hours.

Example 6: Lyophilization of Bacteria

To prepare for the cryoprotectant solution for the bacteria grown in alarge-scale growth condition, saccharose (80 g/L), trehalose (13.3 g/L),sodium glutamate (5.3 g/L), L-cysteine (1.3 g/L) were mixed with water,filter sterilized (0.2 μm filter), and degassed by sparging with aN₂H₂CO₂ (90:5:5) gas. The redox state was monitored with a standardcalibrated redox probe. Degassing continued until the redox valuedropped and stabilized, indicating full anaerobic state (about 60 min).The fully reduced cryoprotectant mix was sealed to protect from airintrusion until use. All Sharples Centrifuge and mixing tank componentswere sterilized in place by autoclaving at 121° C. for 20 min.

The bacteria were grown for 31 hours and harvested from the 20 L or 150L culture using a cooled Sharples Centrifuge set at 10° C. The bacteriain the cylinder was immediately transferred to a sterile blender bag andthe weight of the biomass is measured. An identical weight of thecryoprotectant solution, anaerobic and pre-reduced, was then added tothe blender bag. The anaerobic gas line was inserted into the corner ofthe blender bag. The N₂H₂CO₂ (90:5:5) gas was used to sparge thebacteria and cryoprotectant mix for 5 mins at 6.5 L/min flow rate.

After sparging with anaerobic gas to ensure the concentrated bacteriaand cryoprotectant mix was maintained in an anaerobic condition, theblender bag was sealed placed in a secondary blender bag. Thedouble-walled blender bag was placed inside the JumboMix 3500 paddlemixer and blended for 5 mins on Speed #3 to generate a homogenousanaerobic mix.

After homogenization, the cryoprotectant solution with bacteria waspumped into singe use freeze-drying plates. Each plate is weighed.Plates were immediately transferred to a pre-cooled (−40° C.)lyophilizer for lyophilization using the steps listed in TABLE 8:

TABLE 8 Lyophilization Program Setting. Shelf Ramp temperature time Holdtime Vacuum Procedure Step (° C.) (hour) (hour) (μBar) Freezing 1 −45 05 None Vacuum 2 −45 0 1 400 pull-down Primary 3 −20 5 Wait until the 400drying product temperature is > 25° C. Secondary 4 20 4 24 27 DryingHold 5 22 0.2 Hold 27

The lyophilization procedure finished when the product temperature wasstable for at least two hours. The lyophilized product was ground usinga 1 mm grind setting and stored in vacuum sealed bags at −20° C. untiluse.

Example 7: Comparison of Agar Plating Vs Flow Cytometry for Determiningthe Number of Metabolically Active Cells in a Sample

Two different techniques, conventional agar plating and flow cytometry,were compared for their ability to provide consistent and accuratereadouts on the number of metabolically active cells of a strain (e.g.,strain potency) in a sample, such as a cell suspension or a biologicalsample (e.g., a human fecal sample). Evaluated strains includeAkkermansia muciniphila (DSM 33213), Faecalibacterium prausnitzii (DSM33185), and/or Lactobacillus crispatus (DSM 33187) that can be used in abacterial consortium herein.

In order to determine the number of metabolically active bacterial cells(defined as CFUs) of, e.g., Akkermansia muciniphila (DSM 33213),Faecalibacterium prausnitzii (DSM 33185), and/or Lactobacillus crispatus(DSM 33187) in a sample, the procedure of agar plating was compared tothe use of flow cytometry.

FIGS. 17A-17C show a flow cytometry gating experiment of heat-killedcontrol A. muciniphila (DSM 33213) (A. muciniphila (DSM 33213)) cellsfor quantification of metabolically active therapeutic strains in abacterial cell population, e.g., a cell population that can beadministered to a human subject. Used as an example strain, A.muciniphila (DSM 33213) cell stock solutions were diluted to 10⁻⁴ M in0.9% NaCl buffer solution and placed in a heating block at 95° C. for 20minutes to ensure cell death prior to performing the experiment. Cellswere stained with 2 μM of propidium iodide and 2 μM of SYTO9. A gate isapplied to all cells (FIG. 17A) counted by Forward Scatter Area (FSC-A)and Side Scatter Area (SSC-A) to select for cell size and granularity,respectively. Those cells are then gated on linearity based on ForwardScatter Height (FSC-H) and Forward Scatter-Area (FSC-A) to identifysingle cells (FIG. 17B). The single cells were then used to set gatesfor dead cells (PIhighSYTO9 low) as well as live cells (PI-SYTO9 high),which gives the percentages of live and dead cells in 50 μl of solution(FIG. 17C). FIG. 17A shows flow cytometry results obtained when a gatewas applied to all cells counted by Forward Scatter Area (FSC-A) andSide Scatter Area (SSC-A) to select for cell size and granularity,respectively. FIG. 17B shows flow cytometry results obtained when cellswere then gated on linearity based on Forward Scatter Height (FSC-H) andForward Scatter-Area (FSC-A) to identify single cells. FIG. 17C showsflow cytometry results obtained when single cells were used to set gatesfor dead cells (PIhighSYTO9 low) as well as live cells (PI-SYTO9 high),which gave the percentages of live and dead cells in 50 μL of cellsuspension. This data show that the flow cytometry method allows toaccurately determine the number of metabolically active/inactive cellsas demonstrates in FIG. 17C showing metabolically inactive cells (cellshad been inactivated with heat).

TABLE 9 below shows the sequence of calculations used to calculate thetotal live cells in each dilution. To calculate the “Total CellsCounted”, diluted and unstained cells were counted by the machine andmultiplied by the dilution factor (Dilution Factor X Cells=Total CellsCounted). The “Standard Deviation” and “Average Total Cells” werederived from the “Total Cells Counted”. The “Percent Live Cells” werecalculated by applying the control gates explained in FIGS. 17A-17C tostained cells in dilution. Applying the “Percent Live Cells” to the“Average Total Cells”, the “Total Live Cells” in A. muciniphila (DSM33213) MCB glycerol stock was calculated.

TABLE 9 Calculation of the Number of Live Cells using Flow Cytometry.Percent Dilution Total Cells Standard Average Live Total Live StandardFactor Counted Deviation Total Cells Cells Cells Deviation 0.001 2.21 ×10{circumflex over ( )}10 3.67 × 10{circumflex over ( )}9 1.47 ×10{circumflex over ( )}10 85.2 1.25 × 10{circumflex over ( )}10 1.17 ×10{circumflex over ( )}8 2 1.29 × 10{circumflex over ( )}10 3.67 ×10{circumflex over ( )}9 1.47 × 10{circumflex over ( )}10 84.4 1.24 ×10{circumflex over ( )}10 1.17 × 10{circumflex over ( )}8 4 1.40 ×10{circumflex over ( )}10 3.67 × 10{circumflex over ( )}9 1.47 ×10{circumflex over ( )}10 83.8 1.23 × 10{circumflex over ( )}10 1.17 ×10{circumflex over ( )}8 8 1.42 × 10{circumflex over ( )}10 3.67 ×10{circumflex over ( )}9 1.47 × 10{circumflex over ( )}10 84.1 1.24 ×10{circumflex over ( )}10 1.17 × 10{circumflex over ( )}8 16 1.26 ×10{circumflex over ( )}10 3.67 × 10{circumflex over ( )}9 1.47 ×10{circumflex over ( )}10 82.8 1.22 × 10{circumflex over ( )}10 1.17 ×10{circumflex over ( )}8 32 1.26 × 10{circumflex over ( )}10 3.67 ×10{circumflex over ( )}9 1.47 × 10{circumflex over ( )}10 84.9 1.25 ×10{circumflex over ( )}10 1.17 × 10{circumflex over ( )}8

Similar results were obtained for the strains Faecalibacteriumprausnitzii (DSM 33185), and Lactobacillus crispatus (DSM 33187)demonstrating that flow cytometry can be used to accurately determinethe number of metabolically active therapeutic strains in a sample.Comparing the number of total live cells determined using the platingmethod to those determined by flow cytometry indicates that flowcytometry may allow for a more precise measurement of total live cellsin a sample (see, for example, FIGS. 17A-17C).

In addition, FIG. 18 shows a graph comparing flow cytometryquantification data of live cells with the number of total live cellsdetermined using the (standard) plating method. The data indicate thatflow cytometry allowed a significantly more precise measurement of totallive cells in a sample (e.g., quantified as CFU/mL) compared to theplating method. The left y-axis shows the number of Total Live Cellsmeasured by flow cytometry. The right y-axis shows the calculatedAverage CFU/mL values from nutrient agar plating for biologicalduplicates. A two tailed Mann Whitney t-test showed no significantdifference between the mean values of the two quantification methodswith a p-value of 0.0532. The results show variability of the agarplating technique and the relative consistency of FACS technique,validating the use of the flow cytometer as a method of quantifyingbacterial strains in a biological sample. Samples that may be testedusing this technique include human fecal samples and bacterial strainsamples that may be analyzed for quality control purposes.

Together, these results demonstrate that the flow cytometry methodsdescribed herein can be used determine (i) the ratio of metabolicallyactive to metabolically inactive cells in a sample, and (ii) absolutenumber of metabolically active bacterial cells in a sample.

Example 8: Quantification of Bacterial Cells in Fecal DNA Using qPCR

The use of quantitative polymerase chain reaction (qPCR) is describedfor quantification of the bacterial strains Akkermansia muciniphila (DSM33213), Faecalibacterium prausnitzii (DSM 33185), and Lactobacilluscrispatus (DSM 33187) in a sample. This example describes strainquantification using specific primers, qPCR and fecal DNA as a template.

1. Materials

TABLE 10 below shows exemplary strain-specific primer sequences used forstrain quantification:

TABLE 10 Strain-specific Primer Sequences. SEQ GC Primer Sequence  IDContent (5′ to 3′) NO Strain Tm (%) TATCCGGACTCCTCCATCTG 1A. muciniphila (DSM 33213) 55 62.4 TGTTCGTGCGTTCTTACCTG 2A. muciniphila (DSM 33213) 50 60.4 ATTCCTGAGAAGGCCAGGAT 3A. muciniphila (DSM 33213) 60.4 50 CTGCCGACAAGCATTCCTAT 4A. muciniphila (DSM 33213) 60.4 50 ACCAAGGTTAGCCGCTTTTT 5A. muciniphila (DSM 33213) 45 58.4 CTTGCCCAACAAAATGACCT 6A. muciniphila (DSM 33213) 45 58.4 GTAAGCTCTGTTTCGGCAGCAC 7F. prausnitzii (DSM 33185) 62.3 54.5 ACATTGCACGCTTTGCCGAC 8F. prausnitzii (DSM 33185) 62.7 55 AATTCAGGTTCGGCTGCTGT 9F. prausnitzii (DSM 33185) 60.4 50 CAGGCAGACGTTCTGCTACT 10F. prausnitzii (DSM 33185) 62.4 55 ACGCGTCTCTTTTTGAGCAC 11L. crispatus (DSM 33187) 60.4 50 CCAAATTCAAAGGACTTGGGCT 12L. crispatus (DSM 33187) 60.8 45.45 CGTAGTCCACTTAAGAAGGCCG 13L. crispatus (DSM 33187) 60.73 54.55 GGGCTTTCTTCAAACCTGGC 14L. crispatus (DSM 33187) 59.68 55

PacBio sequencing was performed on genomic DNA extracted from L.crispatus (DSM 33187), F. prausnitzii (DSM 33185), A. muciniphila (DSM33213). Using these data, a comparative genomic analysis of allbacterial strains was performed to identify unique regions within thegenomes of L. crispatus (DSM 33187), F. prausnitzii (DSM 33185), A.muciniphila (DSM 33213). After identification of unique regions, qPCRprimer pairs (see TABLE 10 above) were designed to target the uniqueregions present within these strains.

Additional materials used for this experiment included: (i)A.muciniphila (DSM 33213), F. prausnitzii (DSM 33185), L. crispatus (DSM33187) DNA; (ii) Human Fecal DNA Sample Control (did not containstrains); (iii) extracted DNA from Clinical Samples; (iv) metal 384 qPCRplate holder; (v) DNA/RNAse free, sterile Eppendorf tubes; and (vi)QuantStudio 6 qPCR Thermocycler.

2. Procedure

Clinical sample names and DNA concentrations were calculatedelectronically, and all samples were normalized to 10 ng/μL with a finalvolume of 100 μL. Each run utilized a positive control standard curve of7 points generated with pure bacterial DNA diluted in a human fecal DNAbackground. These standards (abbreviated as “std”) were premade andaliquoted for ease of use. For the standard curve, the following serialdilutions of target strain DNA were included in the run (TABLE 11):

TABLE 11 Standard Strain DNA Preparations. Final DNA Final Amount ofAmount per Well Strain-specific DNA per (including Control StandardReaction well Fecal DNA) Std 1   10 ng 50 ng Std 2    1 ng 50 ng Std 3 0.1 ng 50 ng Std 4 0.01 ng 50 ng Std 5 0.001 ng  50 ng Std 6 0.0001ng   50 ng Std 7 0.00001 ng    50 ngThe following items were used for strain DNA quantification experiments:

TABLE 12 Materials for DNA Quantification Experiments. Item QuantityFecal DNA from Clinical 161 samples (max) Samples (store on ice) A.muciniphila (DSM 33213), ST Strain DNA (Plate Specific) F. prausnitzii(DSM 33185) or L. crispatus (DSM 33187) Standard DNA 384 well plate(sterile) 1 96 well plate (sterile) 2 Eppendorf Tubes (sterile) 3 15 mLFalcon Tube (sterile) 1 Pipette 1000 μL, 200 μL, 10 μL, multichannelpipette Pipette tips (sterile) 1 box of 1000 μL, 200 μL, 10 μL, USP WFI(sterile) 50 mL Tube Rack 1 384 Metal Cooling Plate 1 Styrofoam coolerfull of ice 1 Sealing Film 2 Primers (forward and reverse) 1 stock ofeach strain SYBR Select Master Mix One (1) 5 mL bottle (store on ice)

The calculated amount of water and DNA solution was added to theappropriate wells. The qPCR plate was sealed, vortexed (5-10 seconds)and centrifuged for 2 minutes at 1000 rpm.

3. Preparation of Primer Stocks and Master Mix

The forward and reverse strain specific primers were completely thawed,and primer stocks were maintained at 100 μM in 1×TE buffer (see, forexample, TABLE 10). In two Eppendorf tubes (one for the forward primerand one for the reverse primer) 360 μL of sterile USP grade WFI wasadded. Then, 40 μL of forward primer solution was added and homogenized,and the same step was repeated for the reverse primer solution. Theresulting 10 μM primers solutions were combined with SYBR Select MasterMix (2× Stock) and sterile water and homogenized.

Using the qPCR 384 Well Plate, 20 μL of qPCR Master Mix were aliquotedinto wells A-N 1-24 and O 1-14. After master mix had been aliquoted intoall 350 wells, using the DNA normalization plate, 54 of DNA of all wellsin row A were taken up and inoculated the odd wells in row A in thereaction plate. Then, 5 μL of DNA from all wells in row A wastransferred and inoculated the even wells in row A in the reactionplate. The last 2 steps were repeated for all wells until the DNA wasadded to the reaction plate. Subsequently, the standard curve stock DNAplate of the appropriate strain was transferred to the BiosafetyCabinet, followed by pipetting the standards into the correct wells ofrows P per the reaction plate setup. Following mixing andcentrifugation, the samples were placed in the Quantstudio qPCR machine.

The following cycling conditions were used as shown below in TABLE 13:

TABLE 13 Cycling Conditions. Cycling Conditions Step Temp. ° C. TimeActivation 50 2 min Initial Denaturation 95 2 min Denaturation 95 15seconds 40 Cycles Anneal/Extend 60 1 min

TABLE 14 below shows standard curve control cycle threshold (CT) andprimer melting temperature (TM) values. TABLE 14 further shows, for eachof the selected strains F. prausnitzii (DSM 33185), L. crispatus (DSM33187), and A. muciniphila (DSM 33213), the estimated number of straincells in human fecal DNA with standard amounts of strain cell DNA (e.g.,1 ng, 0.1 ng, 0.01 ng, and 0.001 ng) used for generating a standardcurve that may be used to quantify the amount of strain cell DNA (usinga human fecal DNA background), and the same of amounts of strain cellDNA (e.g., 1 ng, 0.1 ng, 0.01 ng, and 0.001 ng) in water (e.g., withoutfecal DNA

BACKGROUND

TABLE 14 Standard Curve Control Values and Primer TM Values. EstimatedAver- Aver- No. of Strain and Primers age age Sample Name Cells Name CTTM 1 50 ng Human NA F. prausnitzii Primer 03 39.32 83.24 Fecal DNA 50 ngHF DNA + 290000 F. prausnitzii Primer 03 21.51 83.44 1 ng Fp DNA 50 ngHF DNA + 29000 F. prausnitzii Primer 03 23.14 83.34 0.1 ng Fp DNA 50 ngHF DNA + 2900 F. prausnitzii Primer 03 26.04 83.39 0.01 ng Fp DNA 50 ngHF DNA + 290 F. prausnitzii Primer 03 29.67 83.34 0.001 ng Fp DNA Water0 F. prausnitzii Primer 03 NA 61.27 1 ng Fp DNA 290000 F. prausnitziiPrimer 03 21.29 83.64 0.1 ng Fp DNA 29000 F. prausnitzii Primer 03 23.7983.59 0.01 ng Fp DNA 2900 F. prausnitzii Primer 03 26.77 83.54 0.001 ng290 F. prausnitzii Primer 03 30.63 88.32 Fp DNA 50 ng Human NA L.crispatus Primer 02 34.54 73.69 Fecal DNA 50 ng HF DNA + 279000 L.crispatus Primer 02 19.63 73.98 1 ng Lc DNA 50 ng HF DNA + 27900 L.crispatus Primer 02 22.98 74.03 0.1 ng Lc DNA 50 ng HF DNA + 2790 L.crispatus Primer 02 26.15 73.93 0.01 ng Lc DNA 50 ng HF DNA + 279 L.crispatus Primer 02 30.43 73.98 0.001 ng Lc DNA Water 0 L. crispatusPrimer 02 36.00 73.93 1 ng Lc DNA 279000 L. crispatus Primer 02 20.4774.28 0.1 ng Lc DNA 27900 L. crispatus Primer 02 24.01 74.28 0.01 ng LcDNA 2790 L. crispatus Primer 02 26.95 74.23 0.001 ng 279 L. crispatusPrimer 02 30.16 74.08 Lc DNA 50 ng Human NA A. muciniphila Primer 0335.29 77.54 Fecal DNA 50 ng HF DNA + 337000 A. muciniphila Primer 0318.63 77.59 1 ng Am DNA 50 ng HF DNA + 33700 A. muciniphila Primer 0322.01 77.59 0.1 ng Am DNA 50 ng HF DNA + 3370 A. muciniphila Primer 0325.07 77.59 0.01 ng Am DNA 50 ng HF 337 A. muciniphila Primer 03 27.8277.49 DNA + 0.001 ng Am DNA Water 0 A. muciniphila Primer 03 37.85 67.331 ng Am DNA 337000 A. muciniphila Primer 03 20.29 77.78 0.1 ng Am DNA33700 A. muciniphila Primer 03 23.26 77.73 0.01 ng Am 3370 A.muciniphila Primer 03 26.13 77.78 DNA

FIGS. 19A-19C shows limit of detection curves for the three selectedstrains A. muciniphila (DSM 33213), F. prausnitzii (DSM 33185), and L.crispatus (DSM 33187) cells, respectively, that were generated byplotting the measured CT values against the number of estimated straincells as shown in TABLE 14 above.

Example 9: Mouse-Model System

Provided herein is a protocol for assessing the ability of a bacterialconsortium comprising the three bacterial strains L. crispatus (DSM33187), A. muciniphila (DSM 33213), and F. prausnitzii (DSM 33185) forthe treatment of inflammatory diseases, such as allergic diseases, in invivo mouse models.

In an allergic airway inflammation model using young adult micesensitized intratracheally to airway allergen, oral administration ofComposition A, as defined in Example 2, significantly inhibitedelevation of circulating IgE immunoglobulin. In addition, oraladministration of Composition A significantly reduced inflammatory Th2cell expansion in the lung with concurrent reduction in Th2-associatedgene expression and lung concentrations of inflammatory cytokines IL-4and IL-13. Inversely, Composition A resulted in significant expansion ofanti-inflammatory Treg cells in the lungs, which is associated withdecreased allergic asthma-associated molecular and immunologicalresponses. In addition, significant reductions in circulating IgE levelsand airway eosinophilia were observed, while circulating histamine andairway neutrophils trended lower. Based on these studies, and withoutbeing bound to any theory, a proposed mechanism of allergic andasthmatic response mitigation was based on the expansion of Treg cellsand subsequent inhibition of the Th2-driven generation of allergenspecific IgE. It was assumed that expansion of Treg cells was keybecause Composition A administration was also associated with inhibitionof allergic asthma-associated expansion of effector cells, includingeosinophils and neutrophils. In contrast to the monoclonal anti-IgEantibody Xolair® (Omalizumab), which neutralized the IgE effectorantibodies responsible for initiating allergic responses, the mechanismof action using Composition A may have occurred upstream in the cascadeof allergic sensitization highlighting the preventative potential ofComposition A. Composition A was advantageous in that it prevented thegeneration of new IgE and inflammatory effector cells associated withallergic responses and was expected to have far fewer side effects as aresult.

These results demonstrate that the bacterial consortium of Composition Awas effective in treating allergic airway inflammation. It was furtherobserved that the therapeutic efficacy of such consortium may besuperior to that of the monoclonal anti-IgE antibody Xolair®(Omalizumab) while causing significantly fewer side effects. Together,these results can be used as a basis and rationale for a clinical studyin human subjects.

Example 10: Optimized and Ultra-Large-Scale Growth and ManufacturingProcess for Akkermansia muciniphila (DSM 33213) Drug Substance (3500 L)

Manufacturing conditions and procedures for growing Akkermansiamuciniphila (DSM 33213) in 3500 L volume of culture, as outlined in FIG.20, were generated to increase yield and growth rate of bacterialstrains described herein.

Media Preparation

The components for the 250 L sugar feed (TABLE 15), 3160 L of NAGT media(TABLE 16), 1.25 L glacial acetic acid, and cryoprotectant mix (TABLE17) were weighted.

TABLE 15 Recipe for the 250 L sugar feed for A. muciniphila (DSM 33213).Components Weight (kg) for 250 L feed N-acetyl glucosamine 48.8 Dextrose39.8

TABLE 16 Recipe for the 3160 L NAGT Media. Components Weight (kg) for3160 L NAGT media Pea peptone 63.3 Yeast extract 15.8 Sodium chloride0.949 Sodium bicarbonate 3.16 Dibasic potassium 7.91 phosphate Magnesiumsulfate 0.316 heptahydrate Calcium chloride 0.316 L-cysteine HCI 3.16L-threonine 12.7

TABLE 17 Recipe for 100L Cryoprotectant Mix. Weight (kg) for 100Components L cryoprotectant mix Saccharose 8 Trehalose 1.33 SodiumGlutamate 0.55 L-Cysteine HCI 0.13

Sugar Fraction & Feed Preparation and Decontamination

A 300 L container and mixing tank clean in place (CIP) was completed.120 L of hot softened water was added to the sterile mixing tank using a0.22 μm filter. One third of the sugar feed components, by weight, wasadded to the mixing tank and stirred at 150 rpm for 10 minutes untilthey were completely dissolved. Subsequently, the additional two thirdsof sugar feed components and 120 L of hot softened water were added tothe mixing tank and completely dissolved. The sugar feed was filteredsterilized using a 0.2 μm filter and stored in the sterile 300 Lcontainer.

3500 L Culture Media Preparation and Decontamination

To generate the NAGT media, a 3,500 L stirred fermenter is sterilized byCIP and fitted with calibrated pH and redox sensing probes. A total of400 L of 0.22 μm filtered water was added to a presterilized mixing tankand combined with NAGT culture media components. The mixture washomogenized for 10 min at 150 rpm. The concentrated media wastransferred to the 3500 L bioreactor and 3070 L of 0.22 μm filteredsoftened water is added to the fermenter. The pH of the culture mediawas adjusted to pH=6.5. The media was sterilized in place at 121° C. for20 mins. Using a steam sterilized connection, 100 L of the sugar feedwas added to the sterilized NAGT culture medium. The completed NAGTculture medium was then degassed using a sparger adding a N₂H₂CO₂(90:5:5) gas mix at a rate of 0.1 vvm while stirring at 100 rpm andmaintaining a headspace pressure of 0.2 bar. The redox value of the NAGTmedia was monitored from the start of degassing. The degassing continueduntil the redox value dropped and maintained at a steady value for 1hour. The NAGT was then stored at 10° C.±2° C. with 90 RPM stirring andsparging 0.01 vvm of gas mix until use. 20 L and 300 L FermenterPreparation and

Decontamination and Media Transfer

A 20 L and 300 L fermenter clean in place (CIP) was completed. 17 L and300 L of sterile culture media was transferred to the 20 L fermenter and300 L fermenter, respectively, using a sterile connector from the 3500 Lfermenter.

Initial Inoculation Preparation

1 L of sterile NAGT media was transferred from the 20 L fermenter to asterile 1 bottle. The sterile bottle was then transferred to ananaerobic chamber. 19.2 mL of WCB A. muciniphila (DSM 33213) was thawedin the anaerobic chamber and inoculated in the 1 L of reduced NAGT media(2% v/v inoculation rate) using a sterile pipette inoculate. The celland media mixture were homogenized by gently swirling and incubated at37° C. with periodic optical density measurements at 585 nm (OD₆₀₀).FIG. 21 shows that the OD₅₈₅ of the culture rose steadily during thisperiod. The culture was stopped when a) OD₆₀₀>1, or b) if the culturegrew for 48 hours.

20 L Inoculation

20 L culture media was warmed to 37° C. The media was degassed using theparameters listed in TABLE 18.

TABLE 18 Parameters for degassing inoculation. Parameter SettingStirring 100 rpm Temperature 37 ± 2° C. pH 6.5 Gas N₂H₂CO₂ (90:5:5) Gasflow rate 0.01 vvm Overlay 0.2 Bar

The degassing continued until the redox value stabilized for 1 hour, asmeasured by the standard redox sensor. A sterilized 3-way valve wasconnected to the 20 L fermenter. The entire 1 L inoculum culture of A.muciniphila (DSM 33213) (5% v/v inoculum) was added to the 20 Lfermenter via the 3-way valve. The optical density of the 20 L culturewas monitored using OD₅₈₅. FIG. 22 shows that the OD₅₈₅ of the culturerose steadily during this period. The culture was stopped when any oneof the following criteria was reached: a) OD₅₈₅>1.5, b) total culturetime reached 48 hours, or c) a slowing of the growth rate was detectedafter three subsequent OD₅₈₅ readings.

300 L Inoculation

300 L culture media was warmed to 37° C. The media was degassed usingthe parameters listed in TABLE 18. The degassing continued until theredox value stabilized for 1 hour, as measured by the standard redoxsensor. A sterilized 3-way valve was connected to the 300 L fermenter.15 L inoculum culture of A. muciniphila (DSM 33213) (5% v/v inoculum)from the 20 L fermenter was added to the 300 L fermenter via the 3-wayvalve. The optical density of the 20 L culture was monitored usingOD₅₈₅. FIG. 23 shows that the OD₆₀₀ of the culture rose steadily duringthis period. The culture was stopped when any one of the followingcriteria was reached: a) OD₅₈₅>1.5, b) total culture time reached 48hours, or c) a slowing of the growth rate was detected after threesubsequent OD₅₈₅ readings.

3500 L Inoculation

3500 L culture media was warmed to 37° C. The media was degassed usingthe parameters listed in TABLE 18. The degassing continued until theredox value stabilized for 1 hour, as measured by the standard redoxsensor. The 300 L fermenter was connected to the 3500 L fermenter. 300 Linoculum culture of A. muciniphila (DSM 33213) (8-10% v/v inoculum) fromthe 300 L fermenter was added to the 3500 L fermenter via the. Another90 L of the sugar feed was added once glucose concentrations droppedbelow 2 g/L. The optical density of the 20 L culture was monitored usingOD₅₈₅. FIG. 24 shows that the OD₅₈₅ of the culture rose steadily duringthis period. The culture was stopped when any one of the followingcriteria was reached: a) OD_(585 >2.5), b) total culture time reached 72hours, or c) a slowing of the growth rate was detected after threesubsequent OD₅₈₅ readings. When one of these parameters was met, thefermenter was set to 4° C.+/−3° C. to start cooling the culture.

Centrifugation

The GEA centrifuge and mixing tank clean in place (CIP) was completed.The mix gas line (N₂H₂CO₂, 90:5:5) was connected to and degas the GEAcentrifuge and mix tank for 30 mins. The 3500 L culture was centrifugedusing the Sharples parameter listed in TABLE 19.

TABLE 19 Sharples Parameter for the Centrifugation of the 3500 Lculture. Parameter Setting Feed flow rate 600 L/h Counter Pressure 1.5bar

The concentrated bacteria fraction (biomass) was collected in thedegassed mix tank. The weight of the concentrated biomass collected wasweighted.

Cryoprotectant Solution Preparation and Addition to Concentrated Biomass

The mixing tank clean in place (CIP) was completed. 75 L of 0.22 μmfiltered hot softened water was added to the mix tank. One third of thecryoprotectant mix components was added to the mixing tank until theywere completely dissolved. An additional 20 L of softened, filteredwater was added. The mixture was homogenized for 10 min at 150 rpm. Thecryoprotectant solution was transferred to a sterile bioreactor. Thebaseline redox value of the solution was recorded. The cryoprotectantsolution was degassed using the parameters listed in TABLE 20.

TABLE 20 Parameters for degassing the cryoprotectant solution. ParameterSetting Stirring 100 rpm Gas N₂H₂CO₂ (90:5:5) Gas flow rate 0.1 vvmOverlay 0.3 Bar

The degassed cryoprotectant solution was added to the anaerobicconcentrated biomass in the mix tank in a 1:1 (w/w) ratio. The totalmass and volume of biomass and cryoprotectant available forlyophilzation were recorded.

Lyophilization & Grinding

The sterile plastic freeze dry trays were loaded so that the totalthickness does not exceed 1 cm, corresponding to 1.5 L of cell andcryoprotectant mix per tray. The trays were moved into the pre-frozenlyophilizer shelves as they were filled to expedite the freezingprocess. The lyophilization cycle was initiated according to theparameters listed in TABLE 21.

TABLE 21 Parameters for the lyophilization cycle of the concentratedbiomass. Shelf Ramp temperature time Hold time Vacuum Procedure Step (°C.) (hour) (hour) (μBar) Freezing 1 −45 0 3 None Vacuum 2 −45 0 1 400pull-down Primary 3 −5 26 Wait until the 400 drying cell mixturetemperature was > −8° C. Secondary 4 25 4 ≥11.5 27 Drying

The lyophilized material was ground on speed 1, using the spacer 1 and a1 mm grid. After grinding, the lyophilized cell material was immediatelysealed in polyethylene (PE) bags, each containing 1.5 kg or lessmaterial. The lyophilized cell material was stored at <−18° C. and beused to manufacture Composition A, as defined in Example 2.

Example 11: Optimized and Ultra-Large-Scale Growth and ManufacturingProcess for Faecalibacterium prausnitzii (DSM 33185) Drug Substance(3500 L)

Manufacturing conditions and procedures for growing Faecalibacteriumprausnitzii (DSM 33185) in 3500 L volume of culture, as outlined in FIG.25, were generated to increase yield and growth rate of bacterialstrains described herein.

Media Preparation

The components for the 250 L sugar feed (TABLE 22), 3160 L of YFAP media(TABLE 23), and cryoprotectant mix (TABLE 24) were weighted.

TABLE 22 Recipe for the 250 L sugar feed for F. prausnitzii (DSM 33185).Weight (kg) for Components 250 L feed Dextrose 85

TABLE 23 Recipe for the 3160 L YFAP Media. Weight (kg, unless specifiedComponents otherwise) for 3160 L YFAP media Pea peptone 63.3 Yeastextract 15.8 Sodium chloride 3.16 Sodium bicarbonate 3.16 Dibasicpotassium 7.9 phosphate Magnesium sulfate 0.632 heptahydrate Sodiumacetate 15.8 L-cysteine HCI 3.16 Vitamin Mix solution 632 ml

TABLE 24 Recipe for 120L Cryoprotectant Mix. Weight (kg) for 120Components L cryoprotectant mix Saccharose 9.6 Trehalose 1.596 SodiumGlutamate 0.66 L-Cysteine HCI 0.156

Sugar Fraction & Feed Preparation and Decontamination

A 300 L container and mixing tank clean in place (CIP) was completed.120 L of hot softened water was added to the sterile mixing tank using a0.22 μm filter. One third of the sugar feed components, by weight, wasadded to the mixing tank and stirred at 150 rpm for 10 minutes untilthey were completely dissolved. Subsequently, the additional two thirdsof sugar feed components and 130 L of hot softened water were added tothe mixing tank and completely dissolved. The sugar feed was filteredsterilized using a 0.2 μm filter and stored in the sterile 300 Lcontainer.

3500 L Culture Media Preparation and Decontamination

The To generate the YFAP media, a 3,500 L stirred tank bioreactor issterilized by CIP and fitted with calibrated pH and redox sensingprobes. A total of 400 L of 0.22 μm filtered water is added to apre-sterilized mixing tank and combined with YFAP culture mediacomponents and homogenized for 10 min at 150 rpm. The concentrated mediais transferred to the 3500 L bioreactor and 3070 L of 0.22 μm filteredsoftened water is added to the bioreactor. Using a steam sterilizedconnection, 100 L of the sugar feed is added to the sterilized YFAPculture medium. 632 ml Vitamin Mix solution was added. The completedYFAP culture medium is then degassed using a sparger adding a N₂H₂CO₂(90:5:5) gas mix at a rate of 0.1 vvm while stirring at 100 rpm andmaintaining a headspace pressure of 0.2 bar.

Initial Inoculation Preparation

1.6 L of sterile YFAP media was transferred from the 300 L fermenter toa sterile 2 L flask. The sterile bottle was then transferred to ananaerobic chamber. 6.4 mL of WCB F. prausnitzii (DSM 33185) was thawedin the anaerobic chamber and inoculated in the 1 L of reduced YFAP media(0.4% v/v inoculation rate) using a sterile pipette inoculate. The celland media mixture were homogenized by gently swirling and incubated at37° C. with periodic optical density measurements at 585 nm (OD₅₈₅).FIG. 26 shows that the OD₅₈₅ of the culture rose steadily during thisperiod. The culture was stopped when a) OD₅₈₅>3, or b) if the culturegrew for 48 hours.

300 L Inoculation

150 L of sterile culture media was transferred to a sterile 300 Lfermenter The YFAP culture media in the 300 L fermenters was degassedwith a N₂H₂CO₂ (90:5:5) using the sparger for 2 hours at 0.1 vvm 300 Lculture media was warmed to 37° C. The media was degassed using theparameters listed in TABLE 18.

The degassing continued until the redox value stabilized for 1 hour, asmeasured by the standard redox sensor. A sterilized 3-way valve wasconnected to the 300 L fermenter. The entire 1 L inoculum culture of F.prausnitzii (DSM 33185) (2% v/v inoculum) was added to the 20 Lfermenter via the 3-way valve. The optical density of the 20 L culturewas monitored using OD₅₈₅. FIG. 27 shows that the OD₅₈₅ of the culturerose steadily during this period. The culture was stopped when any oneof the following criteria was reached: a) OD₅₈₅>5, b) total culture timereached 48 hours, or c) a slowing of the growth rate was detected afterthree subsequent OD₅₈₅ readings.

3500 L Inoculation

About 3500 L culture media was warmed to 37° C. The media was degassedusing the parameters listed in TABLE 18. The degassing continued untilthe redox value stabilized for 1 hour, as measured by the standard redoxsensor. The 300 L fermenter was connected to the 3500 L fermenter. 30 Linoculum culture of F. prausnitzii (DSM 33185) (8-10% v/v inoculum) fromthe 300 L fermenter was added to the 3500 L fermenter via the. Theoptical density of the 20 L culture was monitored using OD₅₈₅. FIG. 28shows that the OD₅₈₅ of the culture rose steadily during this period.The culture was stopped when any one of the following criteria wasreached: a) OD_(585 >)5, b) total culture time reached 72 hours, or c) aslowing of the growth rate was detected after three subsequent OD₅₈₅readings. When one of these parameters was met, the fermenter was set to4° C.+/−3° C. to start cooling the culture.

Centrifugation

The GEA centrifuge and mixing tank clean in place (CIP) was completed.The mix gas line (N₂H₂CO₂, 90:5:5) was connected to and degas the GEAcentrifuge and mix tank for 30 mins. The 3500 L culture was centrifugedusing the Sharples parameter listed in TABLE 19.

The concentrated bacteria fraction (biomass) was collected in thedegassed mix tank. The weight of the concentrated biomass collected wasweighted.

Cryoprotectant Solution Preparation and Addition to Concentrated Biomass

The mixing tank clean in place (CIP) was completed. 75 L of 0.22 μmfiltered hot softened water was added to the mix tank. One third of thecryoprotectant mix components was added to the mixing tank until theywere completely dissolved. An additional 20 L of softened, filteredwater was added. Subsequently, the additional two thirds ofcryoprotectant components were added to the mixing tank and dissolved.The mixture was homogenized for 10 min at 150 rpm. The cryoprotectantsolution was transferred to a sterile bioreactor. The baseline redoxvalue of the solution was recorded. The cryoprotectant solution wasdegassed using the parameters listed in TABLE 18.

The degassed cryoprotectant solution was added to the anaerobicconcentrated biomass in the mix tank in a 1:1 (w/w) ratio. The totalmass and volume of biomass and cryoprotectant available forlyophilzation were recorded.

Lyophilization & Grinding

The sterile plastic freeze dry trays were loaded so that the totalthickness does not exceed 1 cm, corresponding to 1.5 L of cell andcryoprotectant mix per tray. The trays were moved into the pre-frozenlyophilizer shelves as they were filled to expedite the freezingprocess. The lyophilization cycle was initiated according to theparameters listed in TABLE 21.

The lyophilized material was ground on speed 1, using the spacer 1 and a1 mm grid. After grinding, the lyophilized cell material was immediatelysealed in polyethylene (PE) bags, each containing 1.5 kg or lessmaterial. The lyophilized cell material was stored at <−18° C. and beused to manufacture Composition A, as defined in Example 2.

Example 12: Clinical Study Design

The design of a first in human study is used to evaluate an orallyadministrable pharmaceutical composition comprising a bacterialconsortium consisting of the bacterial strains Akkermansia muciniphila(DSM 33213), Faecalibacterium prausnitzii (DSM 33185), and Lactobacilluscrispatus (DSM 33187) is provided for prevention and treatment ofallergic disease.

The study of this example is designed as a Phase 1b, multi-centered,randomized, double-blind, placebo-controlled, parallel group, threesequential part study of the bacterial consortium consisting of thebacterial strains Akkermansia muciniphila (DSM 33213), Faecalibacteriumprausnitzii (DSM 33185), and Lactobacillus crispatus (DSM 33187) inmulti-sensitized (to two or more allergens) human subjects who areotherwise healthy.

Therapeutic function of bacterial Composition A is evaluated bydetermining change in circulating IgE (both total and specific) levels,immune cell counts, stool microbiome composition, stool and plasmametabolic profile, stool and plasma immune stimulatory capacity(in-vitro analysis), and symptom scores from Baseline to End of theTreatment.

Subjects are separated into 3 categories (see e.g., FIG. 29). The firstcategory contains approximately 20 subjects, 18-40 years of age,multi-sensitized to two or more allergens, who are otherwise healthy(male:female; approximately 1:1) (randomized 3:1 test:Placebo); thesecond category contains approximately 20 subjects, 12-17 years of age,multi-sensitized to two or more allergens, who are otherwise healthy(male:female; approximately 1:1) (randomized 3:1 test:Placebo); and thethird category contains approximately 20 subjects 2-11 years of age,multi-sensitized to two or more allergens, who are otherwise healthy(male:female; approximately 1:1). Subjects are randomized 3:1 withregards to Composition A vs. placebo.

Treatment of human subjects consists of twice daily oral administration(approximately every 12 hours+/−4 hours by mixing with food or milk) ofComposition A for 28 days. Each 1 mL dose of Composition A contains5×10{circumflex over ( )}8 CFU/bacterial species of each Lactobacilluscrispatus (DSM 33187), Akkermansia muciniphila (DSM 33213), andFaecalibacterium prausnitzii (DSM 33185).

Composition A is a live biotherapeutic product containing three livebacterial strains, each present at 5×10{circumflex over ( )}8 CFU perdose: Lactobacillus crispatus (DSM 33187), Faecalibacterium prausnitzii(DSM 33185), and Akkermansia muciniphila (DSM 33213). Composition A issupplied as a single frozen glycerol stock containing all threebacterial species. Each dose of Composition A is provided in a 2 mLpolypropylene screw cap vial with a silicone washer seal. The vialcontains all three live bacterial strains suspended in a bufferedglycerol solution. The buffered glycerol solution is composed ofstandard phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mMNa₂HPO₄, and 1.8 mM KH₂PO₄), 20% v/v glycerol, and 0.1% w/w cysteine asan antioxidant. The volume of the Composition A dose is approximately 1mL.

For the purpose of this example, Composition A is stored at −70° C., anda temperature log is maintained. Once provided to the human subjects,Composition A is stored in a freezer at or below −18° C. Composition Adoses are maintained sealed in its cryovial (e.g., 2 mL cryovial)containers prior to thawing for consumption to maintain the potency andpurity of the product. The frozen stocks of Composition A are thawed atroom temperature for 5-10 minutes and immediately consumed as mentionedin dosing instructions.

The placebo treatment in this example consists of twice daily oraladministration (approximately every 12±4 hours by mixing with food ormilk) of excipients (Placebo: phosphate buffered saline (PBS) asdescribed for product formulation with 20% v/v glycerol and 0.1% w/wcysteine) for 28 days. Each 1 mL dose will be identical in volume to thetest product. The frozen stock is thawed at room temperature for 5-10minutes. After which the contents of the vial are immediately mixed inadequate quantity (1-2 ounces) of cold or room temperature milk(including breast milk, liquid infant formulas, cow's milk, almond milk,and soy milk) or foods such as applesauce and yogurt.

As illustrated in FIG. 29, screening visits are conducted from 28 daysup to 7 days prior to day 1 (baseline visit). Dosing begins on Day 1.Subjects are treated for 28 days with subsequent visits on days 8, 15,22, and 29. Follow-up visits are conducted on days 43 and 57. Each visitafter baseline has a window of ±1 day.

Assessments are performed during the 28 days treatment period and up to28 days (Day 57) of washout thereafter by (i) reporting adverse events;(ii) reporting concomitant medications; (iii) performing physicalexaminations and vital signs; and (iv) laboratory tests including serumchemistry, liver and renal function panels, hematology, complete bloodcount with differential, and urinalysis. In the third part of thisstudy, blood and urine samples are not collected for safety assessments.All other assessments, except those requiring blood and urine sampling,are performed.

Additional assessments are performed by evaluating change from Baselineto End of the Treatment in the following parameters: (i) stoolmicrobiome analysis (bacterial composition); (ii) stool and plasmametabolic profiling; (iii) stool and plasma immune stimulatory capacity(in-vitro analysis); (iv) total IgE and specific IgE levels; (v)circulating immune cell profiling; (vi) questionnaire(s) and adverseeffect (AE) and medication use reporting in diary.

Various laboratory variables are determined in this study. Hematologicalvariables include hematocrit, hemoglobin, mean corpuscular hemoglobin,mean corpuscular hemoglobin concentration, mean corpuscular volume,platelet count, red blood cell distribution width, red blood cell count,and white blood cell count with differential. Urinalysis parametersinclude appearance (e.g., color and character), bilirubin, urobilinogen,protein content, glucose levels, ketones, leukocyte esterase, urineblood, nitrite, pH, and specific gravity. Biochemical parameters includeglucose levels, uric acid, BUN (blood urea nitrogen), creatinine,BUN/creatinine ratio, eGFR (estimated glomerular filtration rate),sodium, potassium, chloride, bicarbonate, calcium, albumin, totalbilirubin, alkaline phosphatase levels, AST (aspartate aminotransferase)levels, ALT (alanine transaminase), gamma-glutamyltransferase (GGT),total cholesterol levels, and triglyceride levels.

Example 13: Viability of Frozen Bacterial Cells

The viability of frozen Akkermansia muciniphila (DSM 33213),Faecalibacterium prausnitzii (DSM 33185), and Lactobacillus crispatus(DSM 33187) in glycerol was examined. About 5×10{circumflex over ( )}8CFUs of A. muciniphila (DSM 33213), about 5×10{circumflex over ( )}8CFUs of F. prausnitzii (DSM 33185), and about 5×10{circumflex over ( )}8CFUs of L. crispatus (DSM 33187) were mixed and frozen in glycerol. Thefrozen bacterial cells were stored in −70° C. The viability of bacterialcells was scored at 1 month, 2 months, 3 months, 6 months, 9 months, and12 months after storage. The score is listed in TABLE 22:

TABLE 22 Viability of Frozen Bacterial Cells in Glycerol Bacteria ColonyForming Unit (CFU) at: Strain 0 month 1 month 2 months 3 months 6 months9 months 12 months A. 4.98 × 10{circumflex over ( )}8 3.19 ×10{circumflex over ( )}8 7.22 × 10{circumflex over ( )}8 1.01 ×10{circumflex over ( )}9 6.78 × 10{circumflex over ( )}8 1.45 ×10{circumflex over ( )}9 2.19 × 10{circumflex over ( )}8 muciniphila(DSM 33213) F. 5.12 × 10{circumflex over ( )}8  5.6 × 10{circumflex over( )}8 5.22 × 10{circumflex over ( )}8 8.06 × 10{circumflex over ( )}86.55 × 10{circumflex over ( )}8 1.65 × 10{circumflex over ( )}9 5.27 ×10{circumflex over ( )}8 prausnitzii (DSM 33185) L. crispatus  5.3 ×10{circumflex over ( )}8   7 × 10{circumflex over ( )}8 6.84 ×10{circumflex over ( )}8 4.59 × 10{circumflex over ( )}8  7.7 ×10{circumflex over ( )}8 6.78 × 10{circumflex over ( )}8 1.75 ×10{circumflex over ( )}9 (DSM 33187)

Example 14: Lyophilized Drug Product Capsule and Liquid CompositionContainer

Composition A, as defined in Example 2, is encompassed in a capsule witha pharmaceutically acceptable excipient. The capsule is in standard size0 or size 1. The capsule is made of plant-derived materials, such astapioca pullulan. It is starch-free, gluten-free, and preservative-free.The capsule is disintegrated at 37° C.>90% of the capsule dissolves inwater, pH=1.2 solution, sodium acetate buffer USP (pH=4.5), or sodiumphosphate buffer (pH=7.2) within 60 minutes. The capsule has adisintegration endpoint of 1.6 minutes, as measured at 37° C. withde-ionized water. The capsule has an oxygen permeability (cm³/m²/day) of≤0.5, as measured by a gas composition in the capsule. The capsule isadministered orally, as shown in FIG. 30. Composition A is also presentin a 2 mL polypropylene screw cap vial and administered orally, as shownin FIG. 30.

Example 15: Clinical Study Design for Preventing Allergic Conditions inInfants and Newborns

Further studies of Composition A: Each dose contains a total of1.5×10{circumflex over ( )}9 CFU, each in equal amounts (5×10{circumflexover ( )}8 CFU/Akkermansia muciniphila (DSM 33213), Faecalibacteriumprausnitzii (DSM 33185), and Lactobacillus crispatus (DSM 33187)) andcarbohydrate-based excipient, compared to placebo (identical-appearingsolution of excipients) in neonate and infant subjects at high risk fordevelopment of allergic diseases. Composition A or placebo isadministered 1 mL once daily for 28 days (Part A) and for 336 days (PartB), orally (by mouth), mixed into breast milk, formula, or food.

In Part A: As illustrated in FIG. 31, Infants aged 28 days to less than12 months are enrolled initially for 28 days of treatment and followedfor 1 additional month observation (off treatment) for evaluation ofsafety/tolerability prior to enrolling neonates (Part B). In Part A,eligible subjects are randomized to either: Composition A, consisting ofonce daily oral administration of Composition A (mixed with breast milk,formula, or food) for 28 days, or placebo, consisting of once daily oraladministration of placebo (mixed with breast milk, formula, or food) for28 days. A total of 20 eligible infants are enrolled in a 3:1 (15:5)randomization.

In Part B: As illustrated in FIG. 32, Neonates of less than or equal to7 days of life are randomized to either Composition A, consisting ofonce daily oral administration of Composition A (mixed with breast milk,formula, or food) for 336 days, or Placebo, a treatment consisting ofonce daily oral administration of placebo (mixed with breast milk,formula, or food) for 336 days.

Eligible neonates are enrolled in a 1:1 (112:112) randomizationstratified by mode of delivery (vaginal or Cesarean section) and bymethod of feeding at enrollment (breast feeding or no breast feeding).Potentially eligible neonates are identified during pregnancy or duringthe first week after birth. Administration of Composition A or placebobegins within 7 days of birth and continue for one year followed by aone-year observation period (off treatment), followed by measurementsduring 10 visits (8 in-clinic visits and 2 telephone calls) over 672days from enrollment.

Immunologic biomarkers, additional stool metabolic profiling, and stoolmicrobiome analysis in Composition A in comparison to placebo in neonateand infant subjects are measured. Therapeutic function of bacterialComposition A is evaluated (secondary endpoints) by determining changein circulating IgE (both total and specific) levels, immune cell counts,stool microbiome composition, stool and plasma metabolic profile, stooland plasma immune stimulatory capacity (in-vitro analysis), and symptomscores from Baseline to End of the Treatment.

The primary endpoint is considered a sensitive PD marker that may beassociated with reduction of development of allergic disease, based onprior studies of the role of live bacterial therapeutics to potentiallymodify the development of allergic disease.

Additional endpoints include: incidence of physician-diagnosed AtopicDermatitis at 168 and 672 days; incidence of physician-diagnosed FoodAllergy, Chronic Rhinitis/Allergic Rhinitis, Urticaria and WheezingIllnesses/Asthma at 168, 336, and 672 days (each diagnosis is assessedindependently); incidence of sensitization to food and aeroallergen at168, 336, and 672 days (as assessed by serum allergen specific IgEtesting to egg white, peanut, cow's milk, cat, house dust mites, dog,Alternaria (mold) and mixed grass pollen); severity of Atopic Dermatitis(as assessed by investigator global assessment (IGA), SCORing AtopicDermatitis (SCORAD) and IGA×BSA at 168, 336, and 672 days); severity ofWheezing Illness/Asthma (assessed by exacerbation history) at 168, 336,and 672 days; concomitant medications prescribed/used for allergicsymptoms or diagnosis and use of rescue medications for atopicdermatitis and wheezing/asthma Total serum IgE levels at 168, 336, and672 days; peripheral eosinophil counts at 168, 336, and 672 days;pharmacogenetics sample (optional); stool microbiome analysis (microbialcomposition); stool and plasma metabolic profiling (selected sitesonly); subcutaneous biomarkers and RNA seq profiling assessed by tapestrips; circulating immune cell profiling (selected sites only); cordblood (biomarker/immune cell profiling) (optional; selected sites only);stool and plasma immune stimulatory capacity (ex-vivo analysis)(selected sites only); titers to tetanus/diphtheria vaccination;severity of Wheezing Illness/Asthma (assessed by Asthma ControlQuestionnaire (ACQ)) at 168, 336, and 672 days.

As a primary study endpoint, safety assessments are performed during the28 days treatment period and up to 28 days (Day 57) of washoutthereafter by (i) reporting adverse events; (ii) reporting concomitantmedications; (iii) performing physical examinations and vital signs; and(iv) laboratory tests including serum chemistry, liver and renalfunction panels, hematology, complete blood count with differential, andurinalysis. In the third part of this study, blood and urine samples arenot collected for safety assessments. All other assessments, exceptthose requiring blood and urine sampling, are performed.

Secondary study assessments are performed by evaluating change fromBaseline to End of the Treatment in the following parameters: (i) stoolmicrobiome analysis (bacterial composition); (ii) stool and plasmametabolic profiling; (iii) stool and plasma immune stimulatory capacity(in-vitro analysis); (iv) total IgE and specific IgE levels; (v)circulating immune cell profiling; (vi) questionnaire(s) and adverseeffect (AE) and medication use reporting in diary.

The strains listed in TABLE 1 herein were deposited at theLeibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH located at Inhoffenstr. 7B, D-38124 Braunschweig, onJun. 27, 2019 in accordance with and under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure. The strains weretested by the DSMZ and determined to be viable. The DSMZ has assignedthe following DSMZ deposit accession numbers to the respective strains:DSM 33213 (A. muciniphila (DSM 33213)), DSM 33179 (B. longum (DSM33179)), DSM 33180 (B. producta (DSM 33180)), DSM 33178 (B.thetaiotaomicron (DSM 33178)), DSM 33176 (C. comes (DSM 33176)), DSM33185 (F. prausnitzii (DSM 33185)), DSM 33191 (F. prausnitzii (DSM33191)), DSM 33186 (F. prausnitzii (DSM 33213)), DSM 33190 (F.prausnitzii (DSM 33190)), DSM 33187 (L. crispatus (DSM 33187)), DSM33177 (B. faecis (DSM 33177)), and DSM 33188 (D. longicatena (DSM33188)).

Additional Embodiments

1. A pharmaceutical composition, wherein the pharmaceutical compositioncomprises: a bacterial consortium, wherein the bacterial consortiumcomprises at least one strain listed in Table 1; and at least oneantioxidant.

2. The pharmaceutical composition of embodiment 1, wherein the bacterialconsortium comprises at least two strains listed in Table 1.

3. The pharmaceutical composition of embodiment 1, wherein the at leastone strain listed in Table 1 comprises A. muciniphila (DSM 33213), F.prausnitzii (DSM 33185), or L. crispatus (DSM 33187).

4. The pharmaceutical composition of embodiment 1, wherein the at leastone strain listed in Table 1 comprises A. muciniphila (DSM 33213), F.prausnitzii (DSM 33185), and L. crispatus (DSM 33187).

5. The pharmaceutical composition of embodiment 1, wherein each strainof the at least one strain listed in Table 1 is present in an amountfrom about 10{circumflex over ( )}7 CFU to about 10{circumflex over( )}9 CFU.

6. The pharmaceutical composition of embodiment 5, wherein the at leastone strain listed in Table 1 is present in an amount of about5×10{circumflex over ( )}8 CFU.

7. The pharmaceutical composition of embodiment 1, wherein the at leastone strain listed in Table 1 is present in a total amount from about10{circumflex over ( )}7 CFU to about 10{circumflex over ( )}10 CFU.

8. The pharmaceutical composition of embodiment 7, wherein the at leastone strain listed in Table 1 is present in an amount of about5×10{circumflex over ( )}9 CFU.

9. The pharmaceutical composition of embodiment 1, wherein the at leastone antioxidant is L-cysteine.

10. The pharmaceutical composition of embodiment 9, wherein theL-cysteine is present in an amount from about 0.05% w/w to about 0.5%w/w.

11. The pharmaceutical composition of embodiment 1, further comprising acryoprotectant.

12. The pharmaceutical composition of embodiment 11, wherein thecryoprotectant is glycerol.

13. The pharmaceutical composition of embodiment 12, wherein theglycerol is present in an amount from about 10% v/v to about 30% v/v.

14. The pharmaceutical composition of embodiment 1, further comprising abuffer.

15. The pharmaceutical composition of embodiment 14, wherein the bufferis PBS.

16. The pharmaceutical composition of embodiment 1, wherein thepharmaceutical composition is formulated in an oral dosage form.

17. The pharmaceutical composition of embodiment 16, wherein the oraldosage form is a capsule, tablet, emulsion, suspension, syrup, gel, gum,paste, herbal tea, drops, dissolving granules, powders, tablets,lyophilizate, a popsicle, or ice cream.

18. The pharmaceutical composition of embodiment 16, wherein the oraldosage form is a suspension.

19. The pharmaceutical composition of embodiment 16, wherein the oraldosage form is a popsicle, or an ice cream.

20. A pharmaceutical composition, wherein the pharmaceutical compositioncomprises: a bacterial mixture, wherein the mixture comprises: A.muciniphila (DSM 33213), F. prausnitzii (DSM 33185), and L. crispatus(DSM 33187); L-cysteine; and a cryoprotectant.

21. The pharmaceutical composition of embodiment 20, wherein each strainof the bacterial mixture is present in amount of about 5×10{circumflexover ( )}8 CFU.

22. The pharmaceutical composition of embodiment 20, wherein theL-cysteine is present in an amount of about 0.1% w/w.

23. The pharmaceutical composition of embodiment 20, wherein thecryoprotectant is glycerol.

24. The pharmaceutical composition of embodiment 23, wherein theglycerol is present in an amount of about 20% v/v.

25. The pharmaceutical composition of embodiment 22, further comprisinga buffer.

26. The pharmaceutical composition of embodiment 25, wherein the bufferis phosphate buffered saline (PBS) and has a pH of about 7.4.

27. The pharmaceutical composition of embodiment 20, wherein thepharmaceutical composition has a total volume of about 1 mL.

28. The pharmaceutical composition of embodiment 20, wherein thepharmaceutical composition is formulated into a suspension for oraladministration.

29. A container comprising a pharmaceutical composition of any one ofembodiments 1-28.

30. The container of embodiment 29, wherein the container is a 2 mLpolypropylene screw cap vial.

31. The container of embodiment 29, wherein the container preserves theviability of at least 95% of bacterial cells in the pharmaceuticalcomposition for about 12 weeks.

32. A kit comprising a container of any one of embodiments 29-31.

33. The kit of embodiment 32, further comprising instructions thatdirect a human user how to use the pharmaceutical composition in thecontainer.

34. A method of manufacturing a pharmaceutical composition of any one ofembodiments 1-28, the method comprising culturing at least two strainsof the pharmaceutical composition, wherein culturing is performed innon-animal media; and combining the products of the culturing stepthereby forming a bacterial consortium.

35. The method of embodiment 34, wherein the non-animal culture mediumis a vegetal culture medium.

36. The method of embodiment 35, wherein vegetal culture mediumcomprises vegetal peptone, vegetal extracts, yeast extract, N-acetylglucosamine (NAG), threonine, or a combination thereof.

37. A method for producing a batch of bacterial cells of a strain ofTable 1, the process comprising growing the bacterial cells in anon-animal culture medium.

38. The method of embodiment 37, wherein the non-animal culture mediumis a vegetal culture medium.

39. The method of embodiment 38, wherein vegetal culture mediumcomprises vegetal peptone, vegetal extracts, yeast extract, N-acetylglucosamine (NAG), threonine, or a combination thereof.

40. The method of embodiment 39, wherein the strain of Table 1 is A.muciniphila (DSM 33213).

41. The method of embodiment 39, wherein the strain of Table 1 is F.prausnitzii (DSM 33185).

42. The method of embodiment 39, wherein the strain of Table 1 is L.crispatus (DSM 33187).

43. A method of manufacturing a pharmaceutical composition comprising abacterial consortium comprising an A. muciniphila strain of Table 1, themethod comprising culturing the Akkermansia sp. cells in a modified NAGTgrowth medium to produce an A. muciniphila cell batch.

44. The method of embodiment 43, wherein the modified NAGT growth mediumcomprises soytone, N-acetyl glucosamine (NAG), or a combination thereof.

45. The method of embodiment 43, wherein the modified NAGT growth mediumdoes not comprise any one or more of magnesium, calcium, or glucose.

46. The method of embodiment 43, wherein the modified NAGT growth mediumprovides a 30-50% increased growth rate of the A. muciniphila cellscompared to unmodified NAGT growth medium.

47. The method of embodiment 46, wherein the increase in growth rate isdetermined by measuring a difference in absorption at 600 nm of thegrowth media after 50 hours of cell culture.

48. The method of embodiment 43, wherein the A. muciniphila strain is A.muciniphila (DSM 33213).

49. The method of embodiment 43, wherein the A. muciniphila cell batchcomprises about 5×10{circumflex over ( )}8 CFU/mL.

50. A method of manufacturing a pharmaceutical composition comprising abacterial consortium comprising Faecalibacterium sp., the methodcomprising culturing the Faecalibacterium sp. cells in anon-animal-based growth medium to produce a Faecalibacterium sp. cellbatch.

51. The method of embodiment 50, wherein the non-animal-based growthmedium is a yeast-based growth medium.

52. The method of embodiment 51, wherein the yeast-based growth mediumcomprises an YFAP vitamin mix.

53. The method of embodiment 51, wherein the yeast-based growth mediumcomprises sodium acetate, soytone, yeast extract, cysteine, or acombination thereof.

54. The method of embodiment 51, wherein the yeast-based growth mediumcomprises sodium acetate, soytone, yeast extract and cysteine.

55. The method of embodiment 50, wherein the Faecalibacterium sp. is F.prausnitzii (DSM 33185).

56. The method of embodiment 50, wherein the Faecalibacterium sp. cellbatch comprises about 5×10{circumflex over ( )}8 CFU/mL.

57. A method of manufacturing a pharmaceutical composition comprising abacterial consortium comprising L. crispatus (DSM 33187), the methodcomprising culturing the L. crispatus (DSM 33187) cells in a yeast-basedgrowth medium to produce a L. crispatus (DSM 33187) cell batch.

58. The method of embodiment 57, wherein the Lactobacillus sp. cellbatch comprises 5×10{circumflex over ( )}8 CFU/mL.

59. A method of treating a disease in a subject, the method comprisingadministering to the subject a pharmaceutical composition of any one ofembodiments 1-27.

60. The method of embodiment 59, wherein the subject is a human.

61. The method of embodiment 60, wherein the human subject is from about18 to about 40 years old.

62. The method of embodiment 60, wherein the human subject is an infantor a neonate.

63. The method of embodiment 62, wherein the infant is from about 0.5years to about 3 years old.

64. The method of embodiment 62, wherein the neonate is no more than 6months old.

65. The method of embodiment 62, wherein the neonate is no more than 3months old.

66. The method of embodiment 57, wherein the pharmaceutical compositionis orally administered to the subject.

67. The method of embodiment 59, wherein the bacterial consortiumconsists of A. muciniphila (DSM 33213), F. prausnitzii (DSM 33185), andL. crispatus (DSM 33187).

68. The method of embodiment 59, wherein the disease is an inflammatorydisease.

69. The method of embodiment 59, wherein the inflammatory disease is anallergy or dermatitis.

70. The method of embodiment 69, wherein the allergy is allergic asthma,allergic pediatric asthma or food allergy.

71. The method of embodiment 59, wherein the disease is a metabolicdisease.

72. The method of embodiment 71, wherein the metabolic disease isobesity, diabetes, or a metabolic syndrome.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is: 1.-22. (canceled)
 23. A method for a growth ofAkkermansia sp. comprising performing a plurality of inoculation roundswith an increasing amount of growth media, wherein an inoculation roundof the plurality of inoculation rounds comprises use of at least about5% by volume of a total batch material of a preceding inoculation roundof the inoculation round of the plurality of inoculation rounds, andwherein a growth media of at least one inoculation round of theplurality of inoculation rounds is at least about 20 liters (L).
 24. Themethod of claim 23, wherein the Akkermansia sp. comprises Akkermansiamuciniphila or Akkermansia glycaniphila.
 25. The method of claim 23,wherein the growth media of the at least one inoculation round of theplurality of inoculation rounds is at least about 150 L.
 26. The methodof claim 23, wherein the plurality of inoculation rounds furthercomprises a second inoculation round, and wherein a growth media of thesecond inoculation round of the plurality of inoculation rounds is fromabout 1 L to about 4,000 L.
 27. The method of claim 23, wherein theplurality of inoculation rounds further comprises an initial inoculationround, wherein the initial inoculation round of the plurality ofinoculation rounds comprises use of a frozen stock of the Akkermansiasp. comprising about 2% by volume of a growth media of the initialinoculation round of the plurality of inoculation rounds.
 28. The methodof claim 23, wherein the plurality of inoculation rounds furthercomprises an initial inoculation round, wherein the initial inoculationround of the plurality of inoculation rounds comprises growing theAkkermansia sp. in an anaerobic condition.
 29. The method of claim 23,wherein the plurality of inoculation rounds further comprises a finalinoculation round, and wherein a growth media of the final inoculationround of the plurality of inoculation rounds is about 3,000 L.
 30. Themethod of claim 29, wherein the final inoculation round of the pluralityof inoculation rounds comprises use of about 10% by volume of a totalbatch material of a preceding inoculation round of the final inoculationround of the plurality of inoculation rounds.
 31. The method of claim23, wherein the at least one inoculation round of the plurality ofinoculation rounds comprises the Akkermansia sp. present in an amount ofOD₅₈₅ of at least 2.5.
 32. The method of claim 23, wherein the precedinginoculation round of the inoculation round is an immediately precedinginoculation round of the inoculation round.
 33. The method of claim 23,further comprising performing a plurality of sterilization rounds and aplurality of degassing rounds for the growth media of the at least oneinoculation round of the plurality of inoculation rounds, wherein asterilization round of the plurality of sterilization rounds comprisesautoclaving the growth media of the at least one inoculation round ofthe plurality of inoculation rounds at 121° C. for 20 minutes, andwherein a degassing round of the plurality of degassing rounds comprisesdegassing the growth media of the at least one inoculation round of theplurality of inoculation rounds with N₂H₂CO₂ (90:5:5).
 34. The method ofclaim 23, further comprising lyophilizing a total batch material of theat least one inoculation round of the plurality of inoculation rounds.35. The method of claim 34, further comprising centrifuging the totalbatch material of the at least one inoculation round of the plurality ofinoculation rounds before the lyophilizing.
 36. The method of claim 34,further comprising grinding the total batch material of at the least oneinoculation round of the plurality of inoculation rounds after thelyophilizing.
 37. The method of claim 23, wherein during a period ofgrowth of a given inoculation round of the plurality of inoculationrounds, a growth media of the given inoculation round of the pluralityof inoculation rounds has a pH value of less than about
 7. 38. Themethod of claim 23, wherein during a period of growth of a giveninoculation round of the plurality of inoculation rounds, a growth mediaof the given inoculation round of the plurality of inoculation roundshas a pH value of less than about 6.5.
 39. A method of forming abacterial composition, comprising: (a) performing a plurality ofinoculation rounds of at least one strain of Akkermansia sp. with anincreasing amount of growth media, wherein an inoculation round of theplurality of inoculation rounds comprises use of at least about 5% byvolume of a total batch material of a preceding inoculation round of theinoculation round of the plurality of inoculation rounds; (b) mixing theat least one strain of Akkermansia sp. with at least one strain ofFaecalibacterium sp.
 40. The method of claim 39, further comprising (c)mixing the at least one strain of Akkermansia sp. with at least onestrain of Lactobacillus sp.
 41. A method of forming a bacterialcomposition, comprising: (a) performing a plurality of inoculationrounds of at least one strain of Akkermansia sp. with an increasingamount of growth media, wherein an inoculation round of the plurality ofinoculation rounds comprises use of at least about 5% by volume of atotal batch material of a preceding inoculation round of the inoculationround of the plurality of inoculation rounds; (b) mixing the at leastone strain of Akkermansia sp. with at least one strain of Lactobacillussp.
 42. The method of claim 41, further comprising (c) mixing the atleast one strain of Akkermansia sp. with at least one strain ofFaecalibacterium sp.